Mutant era and test systems for transactivation

ABSTRACT

The present invention provides in general an artificial cell, an isolated mutant ERα, an isolated polynucleotide encoding the mutant ERα, a method for quantitatively analyzing an activity for transactivation of a reporter gene by a test ERα, a method for screening a mutant ligand dependent transcriptional factor, a method for evaluating an activity for transactivation of a reporter gene by a test ERα, a method for screening a compound useful for treating a disorder of a mutant ERα, a pharmaceutical agent useful for treating an estrogenic disorder of a mutant ERα, use of the mutant ERα, a method for diagnosing a genotype of a polynucleotide encoding a test ERα, a method for diagnosing a genotype of a polynucleotide encoding a test ERα and a method for diagnosing a phenotype of a test ERα.

1. TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to ligand dependent transcriptionalfactors, such as an ERα, and to genes encoding a ligand dependenttranscriptional factor. Further, the present invention relates to cellscontaining a ligand dependent transcriptional factor and a specifiedreporter gene for the ligand dependent transcriptional factor.

2. BACKGROUND OF THE INVENTION

[0002] Various cell mechanisms are regulated by ligand dependenttranscriptional factors. The regulation by the ligand dependenttranscriptional factors is usually achieved because the ligand dependenttranscriptional factor has an activity for transactivation of a gene. Ithas been postulated that in transactivation, the ligand dependenttranscription factor and a RNA polymerase II complex interact togetherat a gene to increase the rate of gene expression. The transactivationcan often determine in eukaryotic cells, whether a gene is sufficientlyexpressed to regulate the various cell mechanisms.

[0003] Such transactivation by the ligand dependent transcriptionalfactors can occur when the ligand dependent tranactivational factor isselectively bound to its cognate ligand and to its cognate responsiveelement sequence. In this regard, the presence of the cognate responsiveelement in a gene or the presence of its cognate ligand in the cell candetermine whether the ligand dependent transcriptional factor cantransactivate the gene.

[0004] ERα is an example of such ligand dependent transcriptionalfactors. ERα is naturally found in the target cells of estrogen such asin ovary cells, breast cells, uterus cells, bone cells and the like. Thetransactivation activity of ERα typically occurs when ERα is selectivelybound to an ERE and an estrogen such as E2. It is reported that aberranttransactivation by ERα may contribute to various disorders. Attemptshave been made to use anti-estrogens that are antagonistic to a normalERα. Examples of such anti-estrogens used with such disorders includetamoxifen, raloxifene, 4-hydroxytamoxifen and the like.

3. SUMMARY OF THE INVENTION

[0005] The present invention provides in general an artificial cell, anisolated mutant ERα, an isolated polynucleotide encoding the mutant ERα,a method for quantitatively analyzing an activity for transactivation ofa reporter gene by a test ERα, a method for screening a mutant liganddependent transcriptional factor, a method for evaluating an activityfor transactivation of a reporter gene by a test ERα, a method forscreening a compound useful for treating a disorder of a mutant ERα, apharmaceutical agent useful for treating an estrogenic disorder of amutant ERα, use of the mutant ERα, a method for diagnosing a genotype ofa polynucleotide encoding a test ERα, a method for diagnosing a genotypeof a polynucleotide encoding a test ERα and a method for diagnosing aphenotype of a test ERα.

4. DESCRIPTION OF FIGURES

[0006] FIGS. 1 to 32 illustrate the luciferase activity provided by ahuman mutant ERα or a human normal ERα. The reporter gene was expressedin the chromosomes of the cell. The mutant ERα gene was transientlyexpressed in the cell. FIGS. 1, 2, 4, 5, 7, 8, 12, 13, 16, 17 and 21 to26 illustrate the luciferase activity in the presence of variousconcentrations of 4-hydroxytamoxifen, raloxifene or ZM189154 as the soleprobable agent of stimulating a human mutant ERα or a human mutant ERα.FIGS. 3, 6, 9 to 11, 14, 15, 18 to 20, 27 to 32 illustrate theluciferase activity in the presence of various concentrations of 100 pMof E2 with various concentrations of 4-hydroxytamoxifen, raloxifene orZM189154. A stable transformed cassette cell was utilized to transientlyexpress the human mutant ERα gene or the human normal ERα gene as wellas to express in a chromosome thereof the reporter gene. FIGS. 1, 2, 4,5, 7, 8, 12, 13, 16, 17 and 21 to 26 zero together the luciferaseactivity by using the luciferase activity provided by the controls, inwhich the human mutant ERα or the human normal ERα was in the presenceof DMSO (containing no 4-hydroxytamoxifen, raloxifene or ZM189154).FIGS. 3, 6, 9-11, 14, 15, 18 to 20, 27 to 32 zero together theluciferase activity by using the luciferase activity provided by thecontrols, in which the human mutant ERα or the human mutant ERα was inthe presence of a DMSO solution containing 100 pM of E2. In zeroing theluciferase activity provided by the human mutant ERα and human normalERα, the luciferase activity by the controls were set together as 100%luciferase activity. The luciferase activity provided by the controls inFIGS. 1, 2, 4, 5, 7, 8, 12, 13, 16, 17 and 21 to 26 is shown as DMSO.The luciferase activity provided by the controls in FIGS. 3, 6, 9-11,14, 15, 18 to 20; 27 to 32 is shown as DMSO+E2.

[0007] FIGS. 33 to 48 illustrate the luciferase activity provided by ahuman mutant ERα or a human normal ERα. The reporter gene, the humanmutant ERα gene and the human normal ERα were expressed in thechromosomes of the cell. FIGS. 33 to 40 illustrate the luciferaseactivity in the presence of various concentrations of4-hydroxytamoxifen, ZM189154 or raloxifene as the sole probable agent ofstimulating a human mutant ERα or a human normal ERα. FIGS. 41 to 48illustrate the luciferase activity in the presence of 100 pM of E2 withvarious concentrations of 4-hydroxytamoxifen, ZM189154 or raloxifene. Astably transformed binary cell was utilized to express in thechromosomes, the reporter gene with the human mutant ERα gene or withthe human normal ERα gene. FIGS. 33 to 40 zero together the luciferaseactivity provided by the controls, in which the human mutant ERα or thehuman normal ERα was in the presence of DMSO (containing no4-hydroxytamoxifen, raloxifene or ZM189154). FIGS. 41 to 48 zerotogether the luciferase activity provided by the controls, in which thehuman mutant ERα or the human normal ERα was in the presence of a DMSOsolution containing 100 pM of E2. In zeroing the luciferase activityprovided by the human mutant ERα and human normal ERα, the luciferaseactivity by the controls were set together as 100% luciferase activity.The luciferase activity provided by the controls in FIGS. 33 to 40 isshown as DMSO. The luciferase activity provided by the controls in FIGS.41 to 48 is shown as DMSO+E2.

[0008] FIGS. 49 to 52 illustrate, as a comparative example, theluciferase activity provided by a human mutant ERαK531E or a humannormal ERα, in which the reporter gene was transiently expressed in thecell. FIGS. 49 and 50 illustrate the luciferase activity in the presenceof various concentrations of 4-hydrozytamoxifen as the sole probableagent of stimulating a human mutant ERα. FIGS. 51 and 52 illustrate theluciferase activity in the presence of 100 pM of E2 with variousconcentrations of 4-hydrxytamoxifen. FIGS. 49 and 50 zero together theluciferase activity of the controls in which the human normal ERα or thehuman mutant ERαK531E was in the presence of DMSO (containing no4-hydrozytamoxifen). FIGS. 51 and 52 zero together the luciferaseactivity of the controls, in which the human normal ERα or the humanmutant ERαK531E was in the presence of a DMSO solution containing 100 pMof E2. In zeroing the luciferase activity provided by the human mutantERα and human normal ERα, the resulting luciferase activity by thecontrols is set as 100% luciferase activity. The luciferase activityprovided by the controls in FIGS. 49 and 50 is shown as DMSO. Theluciferase activity provided by the controls in FIGS. 51 and 52 is shownas DMSO+E2.

5. DETAILED DESCRIPTION OF THE INVENTION

[0009] 5.1. Definitions

[0010] AR: means the androgen receptor protein.

[0011] E2: means estradiol.

[0012] ERα: means an estrogen receptor α protein. Specified mutants ofERα are referred to herein by a letter-number-letter combinationfollowing the phrase “mutant ERα”, such as by K303R, S309F, G390D,M396V, G415V, G494V, K531E and S578P. In the letter-number-lettercombination, the number indicates the relative position of a substitutedamino acid in the mutant ERα, the letter preceding the number indicatesthe amino acid in a normal ERα at the indicated relative position andthe letter following the number indicates the substituted amino acid inthe provided mutant ERα at the indicated relative position. When thereare two substituted amino acids in the mutant ERα, the phrase “mutantERα” is followed by two letter-number-letter combinations, such as byG390D/S578P.

[0013] ERβ: means the estrogen receptor β protein.

[0014] GR: means the glucocorticoid receptor protein.

[0015] MR: means the mineralocorticoid receptor protein.

[0016] PPAR: means the peroxisome proliferator-activated receptorprotein.

[0017] PR: means the progesterone receptor protein.

[0018] PXR: means the pregnane X receptor protein.

[0019] TR: means the thyroid hormone receptor protein.

[0020] VDR: means the vitamin D receptor protein.

[0021] DR1: means the receptor responsive sequence having the followingnucleotide sequence:

[0022] 5′-AGGTCAnAGGTCA-3′

[0023] wherein n represents an A, C, T or G.

[0024] DR3: means the receptor responsive sequence having the followingnucleotide sequence:

[0025] 5′-AGGTCAnnnAGGTCA-3′

[0026] wherein n represents an A, C, T or G.

[0027] DR4: means the receptor responsive sequence having the followingnucleotide sequence:

[0028] 5′-AGGTCAnnnnAGGTCA-3′

[0029] wherein n represents an A, C, T or G.

[0030] ERE: means the estrogen responsive element nucleotide sequence.

[0031] MMTV: means the mouse mammary tumor virus

[0032] 5.2. The Cell

[0033] The cell of the present invention comprises a chromosome whichcomprises a reporter gene. The reporter gene in the chromosome comprisesan ERE, a TATA sequence and a reporter sequence. In addition, the cellcomprises a mutant ERα or a gene encoding the mutant ERα. In thisregard, the cell provides a biological system in which the mutant ERαcan have an activity for transactivation of the reporter gene. Theactivity for transactivation of the reporter gene by the mutant ERα inthe presence of E2 and a partial anti-estrogen is typically higher thanthat by a normal ERα in the presence of E2 and the partialanti-estrogen. Alternatively, the activity for transactivation of thereporter gene by the mutant ERα in the presence of the partialanti-estrogen as the sole probable agent of stimulating the mutant ERαis typically higher than that by the normal ERα in the presence of thepartial anti-estrogen as the sole probable agent of stimulating thenormal ERα.

[0034] Typically, the ERE, the TATA sequence and the reporter sequenceare organized in the reporter gene to allow the transactivation of thereporter gene. For example, the reporter gene can have the ERE operablyupstream from the TATA sequence and the reporter sequence operablydownstream from the TATA sequence. If so desired, the reporter gene mayadditionally contain conventional nucleotide sequences advantageous forthe expression of the reporter gene.

[0035] The TATA sequence may have the following nucleotide sequence:

[0036] 5′-TATAA-3′

[0037] In a natural cell, the ERE is a receptor responsive sequence thatis cognate with a normal ERα. When normal ERα binds to E2 and the normalERα-E2 complex binds to the ERE, the normal ERα has an activity fortransactivation. In the cell, it is a function of the ERE to bind to themutant ERα and allow the mutant ERα to have an activity fortransactivation of the reporter gene. Typically, such an ERE isencompassed by the following nucleotide sequence:

[0038] 5′-AGGTCAnnnTGACCTT-3′

[0039] wherein n represents an A, G, C or T. Further, a tandem repeat ofthe ERE in the reporter gene can provide a more efficient activity fortransactivation of the reporter gene. A 2 to 5 tandem repeat of the EREmay be used in the reporter gene. As an example of an ERE which can beutilized in the reporter gene, there is mentioned an ERE derived fromXenopus vitellogenin gene (Cell, 57, 1139-1146). The ERE can be preparedfor the reporter gene by being chemically synthesized or by being clonedwith polymerase chain reaction (PCR) amplification methods.

[0040] The reporter sequence in the reporter gene is a reporter sequencenaturally foreign to the ERE. As such, the reporter sequence and the EREare not found together in a natural gene. Further, when such a reportersequence encodes a reporter protein, the reporter sequence typicallyencodes a reporter protein that is more or less active in the cell. Asexamples of the reporter protein, there is mentioned a luciferase, asecretory alkaline phosphatase, a β-galactosidase, a chloramphenicolacetyl transferase, a growth hormone and the like.

[0041] Conventional methods may be used to ligate the ERE, the TATAsequence and the reporter sequence. After producing the reporter gene,the reporter gene may be inserted into a chromosome. The reporter genemay be inserted into a chromosome when the reporter gene is introducedinto a host cell. Such methods of introducing the reporter gene into ahost cell are described below.

[0042] The mutant ERα in the cell typically has a particular activityfor transactivation of the reporter gene when in the presence of E2 anda partial anti-estrogen or when in the presence of the partialanti-estrogen as the sole probable agent of stimulating the mutant ERα.The activity for transactivation provided by the mutant ERα in thepresence of E2 and the partial anti-estrogen is typically higher thanthat by a normal ERα in the presence of E2 and the partialanti-estrogen. The activity for transactivation of the reporter gene bythe mutant ERα in the presence of the partial anti-estrogen as the soleprobable agent of stimulating the mutant ERα is higher than that by thenormal ERα in the presence of the partial anti-estrogen as the soleprobable agent of stimulating the normal ERα. Since transactivationinvolves the increase of rate of transcription, such a transactivationby the normal ERα and mutant ERα can be observed by measuring theexpression level of the reporter gene. When the expression levels of thereporter gene provided by the mutant ERα and normal ERα are adjusted tobe zeroed at identical points, the mutant ERα would provide a higherexpression level than that provided by the normal ERα.

[0043] Further, it should be noted that the mutant ERα may have theactivity for transactivation of the reporter gene inhibited in thepresence of the pure anti-estrogen. Such a activity for transactivationfor the reporter gene provided by the mutant ERα is similar to theinhibition of the activity for transactivation of the reporter geneprovided by the normal ERα in the presence of the pure anti-estrogen.

[0044] A normal ERα encompasses the ERα which is reported as mostcommonly carried in a species, such as human, monkey, mouse, rabbit, ratand the like. For example, a human normal ERα has the amino acidsequence shown in SEQ ID: 1. Such a human normal ERα is described inTora L. et al., EMBO, vol 8 no 7: 1981-1986 (1989).

[0045] The partial anti-estrogens typically are not antagonistic to anAF1 region of the normal ERα and are antagonistic to an AF2 region of anormal ERα. The AF2 region of a normal ERα and the AF1 region of anormal ERα are each regions in the normal ERα that are involved intransactivation by the normal ERα (Metzger D. et al., J. Biol. Chem.,270:9535-9542 (1995)).

[0046] Such properties of the partial anti-estrogens may be observed,for example, by carrying out the reporter assay described in Berry M. etal., EMBO J., 9:2811-2818 (1990). In such a reporter assay, there isutilized cells in which the AF1 region of an endogenous normal ERα has astrong activity for transactivation, such as chicken embryo fibroblastcells in primary culture (that may be prepared according to thedescription, for example, in Solomon, J. J., Tissue Cult. Assoc.Manual., 1:7-11 (1975)). When utilized, the chicken embryo fibroblastare modified so that the modified fibroblasts express therein a geneencoding the normal ERα and so that the modified fibroblasts have thereporter gene (hereinafter referred to as AF1 evaluation fibroblasts).When the AF1 evaluation fibroblasts are exposed with a sufficient amountof a partial anti-estrogen, it can be determined whether the partialanti-estrogen fails to be antagonistic to an AF1 region of a normal ERα.The partial anti-estrogen in such cases increase the expression level ofthe reporter gene in the AF1 evaluation fibroblasts. Further, thechicken embryo fibroblast cells in primary culture are then modified fora second round so that the second modified fibroblasts express a geneencoding a truncated normal ERα which has the AF1 region deleted and sothat the second modified fibroblasts have the reporter gene (hereinafterreferred to as AF2 evaluation fibroblasts). When the AF2 evaluationfibroblasts are exposed with a sufficient amount the partialanti-estrogen, it can be determined whether the partial anti-estrogen isantagonistic to an AF2 region of a normal ERα. The partial anti-estrogenin such cases fails to increase the expression level of the reportergene in the AF2 evaluation fibroblasts.

[0047] Examples of such parital anti-estrogens include tamoxifen,4-hydroxytamoxifen, raloxifene and the like.

[0048] The pure anti-estrogen is typically an anti-estrogen which isfully antagonistic to a normal ERα. In this regard, the pureanti-estrogen fails to be partially agonistic to the ERα. In a reporterassay with either the AF1 evaluation fibroblasts or the AF2 evaluationfibroblasts, the pure anti-estrogen provides substantially no activityfor transactivation of the reporter gene by the normal ERα or truncatednormal ERα therein. As such, the expression level of the reporter genein such reporter assays with the pure-anti-estrogen and either of theAF1 evaluation fibroblasts or AF2 evaluation fibroblasts does notsubstantially increase.

[0049] Examples of such pure anti-estrogen include ICI 182780 (WakelingA E et al., Cancer Res., 512:3867-3873 (1991)), ZM 189154 (Dukes M etal., J. Endocrinol., 141:335-341 (1994)) and the like.

[0050] The mutant ERα comprises one or more substituted amino acidswhich confers such an activity for transactivation of the reporter genein the presence of E2 and the partial anti-estrogen or in the presenceof the partial anti-estrogen as the sole probable agent of stimulatingthe mutant ERα. Typically, the one or more substituted amino acids arepresent in the mutant ERα at one or more relative positions of from 303to 578. For example, the mutant ERα may comprise one or more substitutedamino acids at one or more relative positions selected from 303, 309,390, 396, 415, 494, 531, 578 and the like. Typically, such relativepositions in the mutant ERα are based on a homology alignment to theamino acid sequence shown in SEQ ID: 1.

[0051] In general, a homology alignment encompasses an alignment ofamino acid sequences based on the homology of the provided amino acidsequences. For example, Table 1 below randomly sets forth a homologyalignment with the amino acid sequence shown in SEQ ID: 1 (a humannormal ERα), a mouse ERα (Genbank Accession No. M38651), a rat ERα(X6)(Genbank Accession No. X61098) and a rat ERα(Y0) (Genbank Accession No.Y00102). TABLE 1

[0052] In Table 1, “hERa.TXT” sets forth the amino acid sequence shownin SEQ ID: 1, “mER.TXT” sets forth the amino acid sequence of the mouseERα, “ratER(X6).TXT” sets forth the amino acid sequence of the ratERα(X6) and “ratER(Y0)” sets forth the amino acid sequence of ratERα(Y0), wherein amino acids sequences thereof are set forth using oneletter abbreviations of the amino acids. This alignment was preparedusing a commercially available software GENETYX-WIN SV/R ver. 4.0(Software Development Co.). The symbol “*” indicates the amino acidslocated at relative positions 303 and 578.

[0053] The relative positions under the homology alignment correspond tothe absolute positions of the amino acid sequence shown in SEQ ID: 1.For example, relative position 303 encompasses under the homologyalignment, the amino acid in the mutant ERα aligned with the 303rd aminoacid from the N-terminus in the amino acid sequence shown in SEQ ID: 1.Further, a relative position 578 encompasses under the homologyalignment, the amino acid in the mutant ERα aligned with the 578th aminoacid from the N-terminus in the amino acid sequence shown in SEQ ID: 1.In reference to Table 1, examples of relative position 303 include thelysine that is the 303rd amino acid from the amino terminus in the aminoacid sequence shown in SEQ ID: 1, the lysine that is the 307th aminoacid from the amino terminus in the amino acid sequence of the mouseERα, the lysine that is the 308th amino acid from the amino terminus inthe amino acid sequence of rat ERα(X6) and the lysine that is the 308thamino acid from the amino terminus in the amino acid sequence of the ratERα(Y0). Further, examples of the relative position 578 in reference toTable 1 include the serine that is the 578th amino acid from the aminoterminus in the amino acid sequence shown in SEQ ID NO: 1, the serinethat is the 582th amino acid from the amino terminus in the amino acidsequence of the mouse ERα, the serine that is the 583th amino acid fromthe amino terminus in the amino acid sequence of the rat ERα(X6) and theserine that is the 583th amino acid from the amino terminus in the aminoacid sequence of rat ERα(Y0).

[0054] In this regard, the homology alignment in connection with thepresent invention aligns the amino acid sequence shown in SEQ ID: 1 withan amino acid sequence encoding mutant ERα, based on the homology of themutant ERα and the amino acid sequence shown SEQ ID: 1. When aligningthe amino acid sequence of a mutant ERα in the homology alignment toamino acid sequence SEQ ID: 1, such a mutant ERα typically has at leastan 80% homology with the amino acid sequence shown in SEQ ID: 1.

[0055] The mutant ERα can be derived from an animal such as a manual.Examples of such mammals include human, monkey, rabbit, rat, mouse andthe like. For the human mutant ERα, the mutant ERα generally has anamino acid length of 595 amino acids.

[0056] In having the substituted amino acid at relative position 303,the mutant ERα may be derived from changing the lysine present atrelative position 303 in a normal ERα into a substituted amino acid. Insuch cases, the mutant ERα may have the substituted amino acid atrelative position 303 be arginine, such as a mutant ERα K303R. The humanmutant ERαK303R has the amino acid sequence shown in SEQ ID: 2.

[0057] In having the substituted amino acid at relative position 309,the mutant ERα may be derived from changing the serine present atrelative position 309 in a normal ERα into a substituted amino acid. Insuch cases, the mutant ERα may have the substituted amino acid atrelative position 309 be phenylalanine, such as a mutant ERα S309F. Thehuman mutant ERαS309F has the amino acid sequence shown in SEQ ID: 3.

[0058] In having the substituted amino acid at relative position 390,the mutant ERα may be derived from changing the glycine present atrelative position 390 in a normal ERα into a substituted amino acid. Insuch cases, the mutant ERα may have the substituted amino acid atrelative position 390 be aspartic acid, such as a mutant ERα G390D. Thehuman mutant ERαG390D has the amino acid sequence shown in SEQ ID: 4.

[0059] In having the substituted amino acid at relative position 396,the mutant ERα may be derived from changing the methionine present atrelative position 396 in a normal ERα into a substituted amino acid. Insuch cases, the mutant ERα may have the substituted amino acid atrelative position 396 be valine, such as a mutant ERαM396V. The humanmutant ERαM396V has the amino acid sequence shown in SEQ ID: 5.

[0060] In having the substituted amino acid at relative position 415,the mutant ERα may be derived from changing the glycine present atrelative position 415 in a normal ERα into a substituted amino acid. Insuch cases, the mutant ERα may have the substituted amino acid atrelative position 415 be valine, such as a mutant ERαG415V. The humanmutant ERαG415V has the amino acid sequence shown in SEQ ID: 6.

[0061] In having the substituted amino acid at relative position 494,the mutant ERα may be derived from changing the glycine present atrelative position 494 in a normal ERα into a substituted amino acid. Insuch cases, the mutant ERα may have the substituted amino acid atrelative position 494 be valine, such as a mutant ERαG494V. The humanmutant ERαG494V has the amino acid sequence shown in SEQ ID: 7.

[0062] In having the substituted amino acid at relative position 531,the mutant ERα may be derived from changing the lysine present atrelative position 531 in a normal ERα into a substituted amino acid. Insuch cases, the mutant ERα may have the substituted amino acid atrelative position 531 be glutamic acid, such as a mutant ERα K531E. Thehuman mutant ERαK531E has the amino acid sequence shown in SEQ ID: 8.

[0063] In having the substituted amino acid at relative position 578,the mutant ERα may be derived from changing the serine present atrelative position 578 in a normal ERα into a substituted amino acid. Insuch cases, the mutant ERα may have the substituted amino acid atrelative position 578 be proline, such as mutant ERαS578P. The humanmutant ERαS578P has the amino acid sequence shown in SEQ ID: 9.

[0064] In having the substituted amino acid at relative position 390 and578, the mutant ERα may be derived from changing the glycine present atrelative position 390 in a normal ERα into a substituted amino acid aswell as changing the serine present at relative position 578 in thenormal ERα into another substituted amino acid. In such cases, themutant ERα may have the substituted amino acid at relative position 390be aspartic acid and the substituted amino acid at relative position 578be proline, such as mutant ERαG390D/S578P. The human mutantERαG390D/S578P has the amino acid sequence shown in SEQ ID: 10.

[0065] To provide the mutant ERα, the cell may express a gene encodingthe mutant ERα, according to the standard genetic code which is wellknown. Such a mutant ERα gene typically comprises a polynucleotide whichencodes the mutant ERα and a promoter. The mutant ERα gene can beisolated from tissue sample. Further, the mutant ERα gene may beproduced by using mutagenesis techniques to mutagenize a polynucleotideencoding a normal ERα to encode the mutant ERα and by operably linking apromoter upstream from the resulting polynucleotide encoding the mutantERα. The mutagenesis techniques, such as site-directed mutagenesis, maybe utilized to introduce the one or more mutations into the normal ERαpolynucleotide and provide a mutant ERα polynucleotide. The human normalERα polynucleotide having the nucleotide sequence described in Tora L.et al. EMBO J., vol 8 no 7:1981-1986 (1989) is utilized in the case ofmutagenizing the human normal ERα polynucleotide.

[0066] The promoter in the mutant ERα gene initiates transcription sothat the mutant ERα can be expressed to provide the mutant ERα in thecell. In this regard, a promoter capable of functioning in the cell isusually operably linked upstream to a polynucleotide encoding the mutantERα. For instance, where the cell is derived from an animal host cell orfission yeast host cell, examples of the promoter may include Roussarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, early andlate promoters of simian virus (SV40), MMTV promoter and the like. Wherethe cells are derived from budding yeast host cell, examples of thepromoter may include ADH1 promoter and the like.

[0067] In using the mutagenesis techniques, a polynucleotide encodingnormal ERα can be isolated and then the isolated normal ERαpolynucleotide can be mutagenized by using oligonucleotides. Theresulting mutant ERα polynucleotide can then be utilized to produce themutant ERα gene.

[0068] Oligonucleotides are designed and synthesized to specificallyamplify a cDNA encoding a normal ERα from a cDNA library or the cDNAs ofan animal. Such oligonucleotides can be designed, based on a well knownnucleotide sequence encoding the normal ERα, such as the normal ERαnucleotide sequences found in documents, such as Tora L. et al. EMBO J.,vol 8 no 7:1981-1986 (1989), or in databases such as in Genbank. As suchnormal ERα nucleotide sequences, there can be utilized a normal ERαnucleotide sequence derived from human, monkey, rabbit, rat, mouse orthe like. The designed oligonucleotides can then be synthesized with aDNA synthesizer (Model 394, Applied Biosystems). A polymerase chainreaction (PCR) amplification may then be utilized to isolate the normalERα polynucleotide from the cDNA library or cDNAs. For human normal ERαgene, the oligonucleotides depicted in SEQ ID: 11 and SEQ ID: 12 may beutilized to PCR amplify the human normal ERα polynucleotide having thenucleotide sequence described in Tora L. et al. EMBO J., vol 8 no7:1981-1986 (1989).

[0069] The cDNAs can be derived from animal tissue (such as human,monkey, rabbit, rat, or mouse) according to genetic engineeringtechniques described in J. Sambrook, E. F. Frisch, T. Maniatis,“Molecular Cloning, 2nd edition”, Cold Spring Harbor Laboratory, 1989.In such techniques, the RNAs in an animal tissue, such as liver oruterus, are collectively extracted therefrom and the RNAs arecollectively reverse transcribed into the cDNAs of the animal. Forexample, the animal tissue is first homogenized in a buffer containing aprotein denaturing agent such as guanidine hydrochloride or guanidinethiocyanate. Reagents such as a mixture containing phenol and chloroform(hereinafter referred to as phenol-chloroform) are further added todenature proteins resulting from homogenizing the animal tissue. Afterremoving the denatured proteins by centrifugation, the RNAs arecollectively extracted from the recovered supernatant fraction. The RNAscan be collectively extracted by methods such as the guanidinehydrochloride/phenol method, SDS-phenol method, the guanidinethiocyanate/CsCl method and the like. ISOGEN (Nippon Gene) is an exampleof a commercially available kit which is based on such methods ofcollectively extracting the RNAs. After collectively extracting theRNAs, oligo-dT primers are allowed to anneal to the poly A sequence inthe RNAs to collectively reverse transcribe the RNAs as a template. Areverse transcriptase can be utilized to collectively reverse transcribethe RNAs into single-stranded cDNAs. The cDNAs can be synthesized fromthe single-strand cDNAs by using E. coli DNA polymerase I with the abovesingle-stranded cDNAs. In using E. coli DNA polymerase I, E. coli RNaseH is also used to produce primers, which allow E. coli DNA polymerase Ito operate more efficiently. The cDNAs can be purified by usingconventional purifying procedures, for example, by phenol-chloroformextraction and ethanol precipitation. Examples of commercially availablekits based on such methods include cDNA Synthesis System Plus (AmershamPharmacia Biotech), TimeSaver cDNA Synthesis kit (Amerham PharmaciaBiotech) and the like.

[0070] The normal ERα polynucleotide is then isolated from the cDNAs.Isolation procedures which may be utilized to isolate the normal ERαpolynucleotide may include using PCR amplification. The PCRamplification typically amplifies the normal ERα polynucleotide from thecDNAs. The PCR mixture in the PCR amplification may contain a sufficientamount of the cDNAs, a sufficient amount of the forward and reverseoligonucleotides, a heat tolerant DNA polymerase (such as LT-Taqpolymerase (Takara Shuzo)), dNTPs (dATP, dTTP, dGTP, dCTP) and a PCRamplification buffer. In a PCR mixture amplifying a human normal ERαpolynucleotide, there may be utilized 10 ng of the cDNAs and 10 pmol ofthe each of the forward and reverse oligonucleotides (SEQ ID: 11 and SEQID: 12). The PCR mixture in the PCR amplification then undergoes anincubation cycle for annealing, elongation and denaturing. For example,the PCR amplification may have repeated 35 times with a thermal cyclersuch as PCR System 9700 (Applied Biosystems), an incubation cycleentailing an incubation at 95° C. for 1 minute and then an incubation at68° C. for 3 minutes. After the PCR amplification with the cDNAs, thewhole amount of resulting PCR mixture is subjected to low melting pointagarose gel electrophoresis (Agarose L, Nippon Gene). After confirmingthe presence of a band therein comprising the normal ERα polynucleotide,the normal ERα polynucleotide is recovered from the low melting pointagarose gel.

[0071] As the cDNA libraries, there can be utilized a commerciallyavailable cDNA library derived from an animal, such as QUICKClone cDNAs(manufactured by Clontech). The cDNA library may then be isolated asdescribed above.

[0072] The nucleotide sequence of the recovered normal ERαpolynucleotide can be confirmed by preparing a sample of the normal ERαpolynucleotide for direct sequencing. Also, DNA fluorescence sequencingtechniques may be utilized to sequence the normal ERα polynucleotide. Inthis regard, to prepare the sample of the normal ERα polynucleotide,there can be utilized commercially available reagents for fluorescencesequencing such as Dye Terminator Sequencing kit FS (AppliedBiosystems). The fluorescence sequencing of the normal ERαpolynucleotide may be conducted with an autosequencer such as ABIautosequencer (Model 377, Applied Biosystems). Further, the normal ERαpolynucleotide may be manually sequenced (Biotechniques, 7, 494(1989)).

[0073] For convenience, the isolated normal ERα polynucleotide can beinserted into a vector capable of replicating in a host such as E. coli.For example, about 1 μg of isolated normal ERα polynucleotide may havethe ends thereof blunted by a treatment with DNA blunting kit (TakaraShuzo), when the provided isolated normal ERα polynucleotide has unevenends. A T4 polynucleotide kinase may then be used to phosphorylate theends of the blunt-ended normal ERα polynucleotide. After phenoltreatment, the normal ERα polynucleotide is purified by ethanolprecipitation and may be inserted into a vector capable of replicationin E. coli. The E. coli vector comprising the normal ERα polynucleotidemay be cloned into E. coli host cells.

[0074] The E. coli vector comprising the normal ERα polynucleotide maythen be isolated from the cloned E. coli cells. The isolated E. colivector comprising the normal ERα polynucleotide is then used as atemplate to mutagenize, i.e., introduce nucleotide substitutions, intothe normal ERα polynucleotide, such that the resulting mutant ERαpolynucleotide contains a variant codon encoding the substituted aminoacid at the desired relative position.

[0075] The desired nucleotide substitutions may be introduced into thenormal ERα polynucleotide according to the site-directed mutagenesismethods described in J. Sambrook, E. F., Frisch, T. Maniatis, “MolecularCloning 2nd edition”, Cold Spring Harbor Laboratory, 1989, or the sitedirected mutagenesis methods described in McClary J A et al.,Biotechniques 1989(3): 282-289. For example, the desired nucleotidesubstitutions may be introduced into the normal ERα polynucleotide byusing a commercially available kit, such as QuickChange Site-DirectedMutagenesis kit manufactured by Stratagene. Typically, suchsite-directed mutagenesis methods utilize oligonucleotides whichintroduce the desired nucleotide substitutions therein. In relation tothe QuickChange Site-Directed Mutagenesis kit, the following describesin more detail the site-directed mutagenesis methods utilized with thenormal ERα polynucleotide.

[0076] The QuickChange Site-Directed Mutagenesis kit utilizes twooligonucleotides to achieve the desired nucleotide substitution into thenormal ERα polynucleotide. As such a combination of the twooligonucleotides, there may be utilized for human normal ERαpolynucleotide, the combination of oligonucleotides selected from thecombination including the oligonucleotide depicted in SEQ ID: 13 withthe oligonucleotide depicted in SEQ ID: 14, the combination includingthe oligonucleotide depicted in SEQ ID: 15 with the oligonucleotidedepicted in SEQ ID: 16, the combination including the oligonucleotidedepicted in SEQ ID: 17 with the oligonucleotide depicted in SEQ ID: 18,the combination including the oligonucleotide depicted in SEQ ID: 19with the oligonucleotide depicted in SEQ ID: 20, the combinationincluding the oligonucleotide depicted in SEQ ID: 21 with theoligonucleotide depicted in SEQ ID: 22, the combination including theoligonucleotide depicted in SEQ ID: 23 with the oligonucleotide depictedin SEQ ID: 24, the combination including the oligonucleotide depicted inSEQ ID: 25 with the oligonucleotide depicted in SEQ ID: 26 or thecombination including the oligonucleotide depicted in SEQ ID: 27 withthe oligonucleotide depicted in SEQ ID: 28. Table 2 below shows therelative position of the amino acid encoded at the locus of thenucleotide substitution and the resulting variant codons from utilizingsuch combinations of the oligonucleotides. TABLE 2 SEQ ID of oligo-relative nucleotide sequence in variant codon in nucleotides positionencoding normal ERα encoding mutant ERα 13 & 14 303 AAG (lysine) AGG(arginine) 15 & 16 309 TCC (serine) TTC (phenylalanine) 17 & 18 390 GGT(glycine) GAT (aspartic acid) 19 & 20 396 ATG (methionine) GTG (valine)21 & 22 415 GGA (glycine) GTA (valine) 23 & 24 494 GGC (glycine) GTC(valine) 25 & 26 531 AAG (lysine) GAG (glutamic acid) 27 & 28 578 TCC(serine) CCC (proline)

[0077] The achieved mutant ERα polynucleotide can be sequenced toconfirm that the desired nucleotide substitution has been introducedinto the normal ERα polynucleotide.

[0078] To produce the cell, the mutant ERα gene and the reporter geneare usually introduced into a host cell. The reporter gene is introducedinto the host cell so that the reporter gene is inserted into achromosome of the host cell. The mutant ERα gene is introduced into thehost cell for transient expression or is inserted into a chromosome ofthe host cell. When inserting the mutant ERα gene into a chromosome ofthe host cell, the mutant ERα gene and reporter gene may be introducedinto one chromosome or the mutant ERα gene may be inserted intochromosome other than the chromosome utilized for the reporter gene.

[0079] The host cell typically fails have an expressed normal or mutantERα. Examples of the host cells may include budding yeast cells such asCG1945 (Clontech), animal cells such as HeLa cells, CV-1 cells, Hepa1cells, NIH3T3 cells, HepG2 cells, COS1 cells, BF-2 cells, CHH-1 cellsand insect cells and the like.

[0080] The mutant ERα gene and the reporter gene may be inserted intovectors, so that the mutant ERα gene and the reporter gene can beintroduced into the host cell. Such vectors typically have a replicationorigin so that the vector can be replicated in the cell. If so desired,the vector may also have a selective marker gene.

[0081] Where the budding yeast cell is used as a host cell, examples ofthe vector may include plasmid pGBT9, pGAD424, pACT2 (Clontech) and thelike. Where mammalian cells are used as host cells, examples of thevector may include plasmids such as pRc/RSV, pRc/CMV (Invitrogen),vectors containing an autonomous replication origin derived from virusessuch as bovine papilloma virus plasmid pBPV (Amersham PharmaciaBiotech), EB virus plasmid pCEP4 (Invitrogen) and the like.

[0082] When producing a vector encoding the mutant ERα (hereinafterreferred to as the mutant ERα vector), it is preferable for the vectorto additionally contain the promoter so that the mutant ERαpolynucleotide can be inserted into the vector to produce together themutant ERα gene with the mutant ERα vector. Likewise, when producing avector encoding the reporter gene (hereinafter referred to as thereporter vector), it is preferable for the vector to contain a TATAsequence or an ERE so that the reporter gene can be produced togetherwith the reporter vector.

[0083] When producing the mutant ERα vector together with the mutant ERαgene for an animal host cell, pRc/RSV or pRc/CMV can be utilized. Theplasmids pRc/RSV and pRc/CMV contain a promoter which can function inthe cell, when derived from an animal host cell, and a cloning citeoperably downstream from the promoter. In this regard, the mutant ERαvector can be produced together with the mutant ERα gene by insertingthe mutant ERα polynucleotide into pRc/RSV or pRc/CMV at the cloningsite. Since pRc/RSV and pRc/CMV also contain an autonomous replicationorigin of SV40 (ori), pRc/RSV and pRc/CMV may be used to introduce themutant ERα gene into the animal host cells transformed with ori(−) SV40genome, if so desired. As such animal host cells transformed with ori(−)SV40 genome, there is mentioned COS cells. When introduced into suchanimal host cells transformed with ori(−) SV40 genome, the mutant ERαvector produced from pRc/RSV or pRc/CMV can increase to a fairly largecopy number therein such that the mutant ERα gene can be expressed in alarge amount.

[0084] When introducing the mutant ERα vector into a budding yeast hostcell, it is preferable to utilize pACT2 to produce the mutant ERαvector. Since pACT2 carries an ADH1 promoter, the mutant ERα gene can beproduced together with the mutant ERα vector by inserting the mutant ERαpolynucleotide downstream of the ADH1 promoter. In such cases, a themutant ERα vector can express the mutant ERα gene in a large amount.

[0085] Conventional techniques can be used for introducing the mutantERα gene, according to the type of host cell. For example, the calciumphosphate method, DEAE-dextran method, electroporation, lipofection orthe like may be use where mammalian or insect cells are used as hostcells. Where yeast cells are used as host cells, there may be used alithium method such as a method using the Yeast transformation kit(Clontech) or the like.

[0086] Futhermore, where the mutant ERα gene is introduced into the hostcell as viral DNA, the mutant ERα gene may be introduced into host cellsnot only by the techniques as described above, but also by infecting thehost cells with recombinant virions containing the vital forms of thereporter gene and the mutant ERα gene. For example, viruses such asvaccinia virus may utilized for animal host cells and where insectanimal cells are used as host cells, there may be utilized insectviruses such as baculovirus.

[0087] When the mutant ERα vector or the reporter vector comprises theselective marker gene, as described above, the selective marker gene maybe employed to clone the cell of the present invention. In such cases,the selective marker gene can be utilized to confer a drug resistance toa selective drug exhibiting lethal activity on the cell. The cell inthis regard may then be cloned by culturing the cell in a mediumsupplemented with said selective drug. Exemplary combinations of theselective marker gene and selective drug include a combination ofneomycin resistance-conferring selective marker gene and neomycin, acombination of hygromycin resistance-conferring selective marker geneand hygromycin, a combination of blasticidin S resistance-conferringselective marker gene and blasticidin S and the like. In a case whereinthe selection marker gene encodes a nutrient which complements theauxotrophic properties of the cell, the cell may be cultured using aminimal medium that substantially contains none of the nutrient.Furthermore, an assay measuring an estrogen binding activity may alsoused to clone the cell.

[0088] In introducing the reporter gene into the host cell, the reportergene is usually introduced in a linearized form. The linearized reportergene may allow the reporter gene to be inserted into the chromosome ofthe host cell. When utilizing the reporter vector, the reporter vectorcan be linearized by a restriction digestion. The lipofection method maybe utilized to introduce the linearized reporter gene into the hostcell.

[0089] Further, it should be noted that the reporter gene may beintroduced into the host cell, before introducing the mutant ERα gene toprovide a stably transformed cassette cell. The stably transformedcassette cell stably comprises the reporter gene in a chromosome thereofsuch that the reporter gene can be genetically handed down to progenygenerations. To produce the stably transformed cassette cell, thereporter gene may be introduced into the chromosome of a host cell andthe host cell may be cultured for several weeks. After culturing forseveral weeks, the stably transformed cassette cell can be cloned byemploying the selective marker gene, when utilized. For example, thetransformed host cells may be continuously cultured for several weeks ina medium supplemented with the selective drug to clone the stabletransformed cassette cell. The mutant ERα gene may then be introducedinto the stably transformed cassette cell to produce the cell.

[0090] Furthermore, the mutant ERα gene may also be introduced into thehost cell with the reporter gene so that the host cell is stablytransformed with the reporter gene and the mutant ERα gene.

[0091] The cell can be utilized to screen for a compound useful fortreating a disorder of the mutant ERα. Such a disorder of a mutant ERαmay be a disorder which involves an aberrant transactivation by themutant ERα, such as breast cancer. To screen such a compound, the cellis exposed with an efficient amount of a test compound suspected ofbeing antagonistic or agonistic to the mutant ERα and thetransactivation level of the reporter gene is measured.

[0092] The cell is typically exposed with a sufficient amount of thetest compound for one to several days. The cell can be exposed with thetest compound under agonistic conditions or antagonistic conditionsdirected to the mutant ERα. The agonistic conditions typically have theassay cell exposed to the test compound as the sole agent probable ofstimulating the mutant ERα. The antagonistic conditions typically havethe assay cell exposed to the test compound and E2.

[0093] After exposure, the transactivation level of the reporter gene ismeasured by measuring the expression level of the reporter gene. In suchcases, the reporter protein or the reporter RNA (encoded by the reportersequence) is stored in the cell or is secreted from the cell so that theexpression level can be measured therewith. The expression level of thereporter gene can be measured by a Northern blot analysis, by a Westernblot analysis or by measuring the activity level of the reporterprotein. The activity level of the reporter protein typically indicatesthe level at which the reporter gene is expressed.

[0094] For example, when the reporter gene encodes luciferase as thereporter protein, the expression level of the reporter gene can bemeasured by the luminescence provided by reacting luciferin andluciferase. In such cases, a crude cell extract is produced from thecells and luciferin is added to the crude cell extract. The luciferinmay be allowed to react with the luciferase in the cell extract at roomtemperature. The luminescence from adding luciferin is usually measuredas an indicator of the expression level or the reporter gene, since thecrude cell extract produces a luminescence at a strength proportional tothe level of luciferase expressed in the cell and present in the crudecell extract. A luminometer may be utilized to measure the luminescencein the resulting crude cell extract.

[0095] The measured transactivation level can then be compared with acontrol to evaluate the agonistic or antagonistic effect of the testcompound. Such a control in screening the test compound can be theexpected transactivation level of the reporter gene when the cell is notexposed to the test compound. When the transactivation level of thereporter gene by the mutant ERα is higher than the control under theagonistic conditions, the test compound is evaluated as an agonistdirected to the mutant ERα.

[0096] Alternatively, when the cell is exposed to E2 and the testcompound under the antagonistic conditions, the test compound can beevaluated as an antagonist directed to the mutant ERα. In such cases,the control can be the expected transactivation level of the reportergene by the mutant ERα in the presence of an equivalent amount of E2.When the transactivation level of the reporter gene by the mutant ERα islower than the control, the test compound is evaluated as beingantagonistic to the mutant ERα.

[0097] Such a test compound agonistic or antagonistic to the mutant ERαcan then be selected as a compound useful for treating a disorder of themutant ERα. In such cases, the test compound which provides antransactivation level of the reporter gene which is significantly higherthan the control is usually selected when the cell is exposed under theagonistic conditions. The test compound which provides antransactivation level of the reporter gene which is significantly lowerthan the control is usually selected when the cell is exposed under theantagonistic conditions.

[0098] Futhermore, compounds for treating disorders of normal liganddependent transcription factors can be screened. In such cases, a geneencoding the normal ligand dependent transcription factor, instead ofthe mutant ERα gene, is introduced into the host cell. Examples of suchnormal ligand dependent transcription factors include a normal ERβ(Genbank Accession No. AB006590), a normal AR (Genbank Accession No.M23263), a normal GR (Genbank Accession No. M10901), a normal TRα(M24748), a normal PR (Genbank Accession No. 15716), a normal PXR(Genbank Accession No. AF061056), a normal lipophilic vitamin receptorsuch as a normal VDR (Genbank Accession No. J03258), a normal RAR(Genbank Accession No. 06538), a normal MR (Genbank Accession No.M16801), a normal PPAR γ (Genbank Accession No. U79012) and the like.The reporter gene in such cases comprises an appropriate receptorresponsive sequence cognate with the normal ligand dependenttranscriptional factor, instead of the ERE.

[0099] 5.3. The Diagnosis Methods

[0100] The diagnosis methods of the present invention involve diagnosingthe phenotype of a test ERα or the genotype of a polynucleotide encodingthe test ERα. In the genotype diagnosis methods, it can be determinedwhether the polynucleotide encoding the test ERα contains a valiantcodon therein which provides for the one or more substituted amino acidswhich confer the activity for transactivation of the reporter gene, asdescribed in the above 5.2. In the phenotype diagnosis methods, it canbe determined whether the test ERα contains one or more substitutedamino acids therein which confer the activity for transactivation of thereporter gene as described in the above 5.2.

[0101] The genotype diagnosis methods typically involve preparing thetest ERα polynucleotide, searching for the variant codon and determiningthe mutation in the variant codon, if present. Examples of such genotypediagnosis methods include PCR amplification and nucleotide sequencingmethods, single strand conformation polymorphism (SSCP) methods,restriction fragment length polymorphism (RFLP) methods, hybridizationmethods and the like.

[0102] The test ERα polynucleotide can be prepared for the genotypediagnosis methods by preparing test genomic DNAs or test cDNA. In suchcases, test genomic DNAs or test cDNAs, which contain the test ERαpolynucleotide, are collectively prepared from a test sample obtainedfrom a test animal, such as a test human. Such a test sample may beobtained from non-surgical methods, from surgical methods such as from afine needle or from a biopsy or the like. Examples of such test samplesinclude the cellular tissue of the test mammal, such as hair, peripheralblood, oral epithelial tissue, liver, prostate, ovaries, uterus, mammarygland or the like, from which test genomic DNAs or test cDNAs can beextracted.

[0103] For example, the test genomic DNAs can be prepared according tothe methods described in TAKARA PCR Technical news No. 2 (Takara Shuzo,1991.9). In such cases, a test sample of 2 to 3 hairs from a test mammalare washed with sterile water and ethanol and are cut into 2 to 3 mm inlength. The test cells in the hairs are then lysed with a sufficientamount, such as 200 μl, of BCL buffer (10 mM of Tris-HCl (pH 7.5), 5 mMof MgCl₂, 0.32 M of sucrose, 1% of Triton X-100). The test genomic DNAstherefrom are washed from unnecessary proteins by adding and mixingProteinase K and SDS to the lysed test cells to amount to finalconcentrations of 100 μl/ml and 0.5% (w/v), respectively. Afterincubating the reaction mixture at 70° C., the test genomic DNAs can bepurified by a phenol-chloroform extraction.

[0104] Additionally, when the test sample is peripheral blood, testgenomic DNAs can be collectively obtained, for example, by processingthe test sample with DNA-Extraction kit (Stratagene).

[0105] Also, when the test sample is obtained from a biopsy, the testcDNAs may be prepared from the test sample by collectively reversetranscribing the RNAs in the cellular tissue. The RNAs can becollectively obtained from the cellular tissue by using TRIZOL reagent(Gibco), and preferably when the cellular tissue is still fresh.

[0106] Furthermore, the test genomic DNAs can be prepared according tothe methods described in M. Muramatsu “Labo-Manual-Idenshi-Kogaku”(Maruzen, 1988).

[0107] Even furthermore, the test cDNAs may be prepared according to thegenetic engineering techniques described in J. Sambrook, E. F. Frisch,T. Maniatis, “Molecular Cloning 2nd edition”, Cold Spring HarborLaboratory, 1989, as described in the above 5.2.

[0108] When searching for the variant codon in the genotype diagnosismethods, a searching region in the test ERα polynucleotide typicallyincludes codons therein which are suspected to be the variant codon. Assuch, the searching region in the test ERα polynucleotide may includethe codons in the test ERα polynucleotide which encode the amino acidsin the test ERα at relative positions 303 to 578. For example, suchgenotype diagnosis methods may have the searching region include a codonin the test ERα polynucleotide which encode an amino acid at relativepositions selected from 303, 309, 390, 396, 415, 494, 531, 578 and thelike.

[0109] The PCR amplification and sequencing methods as well as the SSCPmethods may then use the prepared test cDNAs or the test genomic DNAs tospecifically PCR amplify the searching regions in the test ERαpolynucleotide therefrom. Search oligonucleotides can be utilized tospecifically PCR amplify from the test cDNAs or test genomic DNAs, thesearching regions present in the test ERα polynucleotide.

[0110] The search oligonucleotides in this PCR amplification aretypically designed to specifically PCR amplify the searching region inthe test ERα polynucleotide. The search oligonucleotides may have a sizeof from 8 to 50 bp, preferably 15 to 40 bp, and may have a GC content of30% to 70%. Such search oligonucleotides may be synthesized with a DNAsynthesizer using the β-cyanoethyl phosphoamnidide methods,thiophosphite methods or the like. Further, the search oligonucleotidesmay be unlabeled, non-radioactively labeled, radiolabeled such as with³²P or the like. The PCR amplification typically utilizes a combinationof a forward search oligonucleotide and a reverse search oligonucleotideto specifically PCR amplify the searching region in the test ERαpolynucleotide. Examples of such combinations of forward and reversesearch oligonucleotides for a human test ERα polynucleotide are shownbelow in Table 3, in connection with the relative position of the aminoacid encoded in the searching region. TABLE 3 SEQ IDs depicting thesearch oligonucleotides relative Forward search oligonucleotide Reversesearch oligonucleotide position SEQ ID:29, SEQ ID:30, SEQ ID:31, SEQID:34, SEQ ID:35, SEQ ID:36, 303 SEQ ID:32 or SEQ ID:33 SEQ ID:37 or SEQID:38 SEQ ID:39, SEQ ID: 40, SEQ ID:41, SEQ ID:44, SEQ ID: 45, SEQID:46, 309 SEQ ID:42 or SEQ ID:43 SEQ ID:47 or SEQ ID:48 SEQ ID:49, SEQID: 50, SEQ ID:51, SEQ ID:54, SEQ ID: 55, SEQ ID:56, 390 SEQ ID:52 orSEQ ID:53 SEQ ID:57 or SEQ ID:58 SEQ ID:59, SEQ ID: 60, SEQ ID:61, SEQID:64, SEQ ID: 65, SEQ ID:66, 396 SEQ ID:62 or SEQ ID:63 SEQ ID:67 orSEQ ID:68 SEQ ID:69, SEQ ID: 70, SEQ ID:71, SEQ ID:74, SEQ ID: 75, SEQID:76, 415 SEQ ID:72 or SEQ ID:73 SEQ ID:77 or SEQ ID:78 SEQ ID:79, SEQID: 80, SEQ ID:81, SEQ ID:84, SEQ ID: 85, SEQ ID:86, 494 SEQ ID:82 orSEQ ID:83 SEQ ID:87 or SEQ ID:88 SEQ ID:89, SEQ ID: 90, SEQ ID:91, SEQID:94, SEQ ID: 95, SEQ ID:96, 531 SEQ ID:92 or SEQ ID:93 SEQ ID:97 orSEQ ID:98 SEQ ID:99, SEQ ID: 100, SEQ SEQ ID:104, SEQ ID: 105, SEQ 578ID:101, SEQ ID:102 or SEQ ID:103 ID:106, SEQ ID:107 or SEQ ID:108

[0111] The searching regions in the test ERα polynucleotide can bespecifically PCR amplified from the test cDNAs or the test genomic DNAsaccording to the methods described in Saiki et al., Science, vol. 230,pp. 1350-1354 (1985). The PCR mixture in this PCR amplification maycontain 1.5 mM to 3.0 mM magnesium chloride, heat tolerant DNApolymerase, dNTPs (dATP, dTTP, dGTP, and dCTP), one of the forwardsearch oligonucleotides in combination with one of the reverse searcholigonucleotides and the test genomic DNAs or test cDNAs. In this PCRamplification, there may be repeated 20 to 50 times, preferably 25 to 40times, an incubation cycle entailing a denaturation incubation, anannealing incubation and an elongation incubation. The denaturationincubation may incubate the PCR mixture at 90° C. to 95° C., andpreferably at 94° C. to 95° C., for 1 min to 5 min, and preferably for 1min to 2 min. The annealing incubation following the denaturingincubation may incubate the PCR mixture at 30° C. to 70° C., andpreferably at 40° C. to 60° C., for 3 seconds to 3 minutes, andpreferably for 5 seconds to 2 minutes. The elongation incubationfollowing the denaturing incubation may incubate the PCR mixture at 70°C. to 75° C., and preferably at 72° C. to 73° C., for about 15 secondsto 5 minutes, and preferably for 30 seconds to 4 minutes.

[0112] When utilizing the PCR amplification and nucleotide sequencingmethods, the genotype diagnosis methods may then entail subjecting theresulting PCR mixture to low melting point agarose gel electrophoresis.The amplified polynucleotide encoding the searching region (hereinafterreferred to as searching region polynucleotide) is recovered from thelow melting point agarose gel and is sequenced to provide a nucleotidesequence of the searching region polynucleotide.

[0113] The mutation in the variant codon, if present, may then bedetermined by sequencing the searching region polynucleotide and bydetermining the mutation in the nucleotide sequence. In sequencing thesearching region polynucleotide, there may be utilized the directsequencing methods or an automated sequencing method. Examples of thedirect sequencing methods include manual sequencing methods (MaxamGilbert method described in Maxam, A. M. & W. Gilbert, Proc. Natl. Acad.Sci. USA, 74, 560, 1977), the Sanger method (described in Sanger, F. &A. R. Coulson, J. Mol. Biol., 94, 441, 1975 as well as Sanger, F.,Nicklen, and A. R., Coulson, Proc. Natl. Acad. Sci. USA., 74, 5463,1977), the methods described in BioTechniques, 7, 494 (1989) and thelike. When an automated DNA sequencer such as ABI autosequencer (Model377.Applied Biosystems) is used, an appropriate DNA sequencing kit suchas ABI Big Dye terminator cycle sequencing ready reaction kit can beused to prepared the searching region for the automated DNA sequencer.After sequencing, the nucleotide sequence of the searching regionpolynucleotide may then be compared to a nucleotide sequence encoding anormal ERα to determine the mutation in the valiant codon, if present,in the searching region.

[0114] When utilizing the SSCP methods, the resulting PCR mixture issubjected to a native polyacrylamide gel electrophoresis according tothe methods described in Hum. Mutation, vol. 2, p. 338. In such cases,it is preferable that the PCR amplification above utilize theradiolabeled oligonucleotides so that the searching regionpolynucleotide is radiolabeled and the searching region polynucleotidecan be detected in the native polyacrylamide gel by employing theradioactivity thereof. In such SSCP methods, the radiolabeled searchingregion polynucleotide can be heat-denatured into single strandpolynucleotides and subjected to the native polyacrylamide gelelectrophoresis in a buffer to separate each of the single strandpolynucleotides. Examples of buffers which may be utilized in the nativepolyacrylamide gel electrophoresis include Tris-phosphate (pH 7.5-8.0),Tris-acetate (pH 7.5-8.0), Tris-borate (pH 7.5-8.3) and the like, withTris-borate (pH 7.5-8.3) being preferred. In addition, auxiliarycomponents for the native polyacrylamide gel electrophoresis may beutilized in the buffer, such as EDTA. The conditions for such nativepolyacrylamide gel electrophoresis may include a constant power of 30 to40 W at 4° C. to room temperature (about 20 to 25° C.) for 1 hour to 4hours.

[0115] After the native polyacrylamide gel electrophoresis, the nativepolyacrylamide gel is transferred onto a filter paper and contacted withX-ray film to expose the X-ray film with the radiation from theradiolabeled searching region polynucleotide. An appropriate cassettemay be utilized to expose the X-ray film. The autoradiogram obtainedfrom developing the X-ray film allows a comparison of the mobility ofthe radiolabeled searching region polynucleotide with the mobility of astandard. Such a mobility of the standard can be the mobility expectedwhen the searching region polynucleotide is composed of only normalcodons of the normal ERα polynucleotide. A mobility of the radiolabeledsearching region polynucleotide different from the mobility of thestandard typically indicates that there is one or more valiant codons inthe radiolabeled searching region.

[0116] The radiolabeled searching region polynucleotide may then berecovered from the native polyacrylamide gel by using heated or boilingwater. The radiolabeled searching region may be PCR amplified for asecond round and then prepared for sequencing. The mutation in thevariant codon, if present, may then be determined similarly to themethods described above in the PCR amplification and nucleotidesequencing methods.

[0117] The hybridization methods typically utilize a probeoligonucleotide to observe whether the probe oligonucleotide canhybridize to the searching regions. The searching regions can beprovided in the hybridization methods by utilizing the searching regionpolynucleotide, the prepared test cDNA, the prepared test genonic DNA, apurified test ERα polynucleotide or the like. Further, the hybridizationmethods may restriction digest the searching region polynucleotide andthen utilize the restriction digested searching region polynucleotide toobserve whether the probe oligonucleotide can hybridize thereto.

[0118] The probe oligonucleotides may have a size of from 15 to 40 bp,and may have a GC content of 30% to 70%. Such probe oligonucleotides maybe synthesized with a DNA synthesizer using the β-cyanoethylphosphoamidide methods, thiophosphite methods or the like. Further, theprobe oligonucleotides are typically non-radioactively labeled such aswith biotin, radiolabeled such as with ³²P or the like.

[0119] The probe oligonucleotides may be composed of the nucleotidesequence of the searching region, when the searching region is composedof only normal codons of a normal ERα polynucleotide. Such a nucleotidesequence allows the probe oligonucleotides to hybridize to the searchingregion in the test ERα polynucleotide under stringent conditions, whenthe searching region therein is composed of only normal codons of anormal ERα polynucleotide. Examples of such probe oligonucleotides for ahuman test ERα polynucleotide are shown below in Table 3, in connectionwith the relative position of the amino acid encoded in the searchingregion. TABLE 3 Probe oligonucleotide relative position SEQ ID:111, SEQID:112, SEQ 303 ID:113, SEQ ID:114 or SEQ ID:115 SEQ ID:116, SEQ ID:117, SEQ 309 ID:118, SEQ ID:119 or SEQ ID:120 SEQ ID:121, SEQ ID: 122,SEQ 390 ID:123, SEQ ID:124 or SEQ ID:125 SEQ ID:126, SEQ ID: 127, SEQ396 ID:128, SEQ ID:129 or SEQ ID:130 SEQ ID:131, SEQ ID: 132, SEQ 415ID:133, SEQ ID:134 or SEQ ID:135 SEQ ID:136, SEQ ID: 137, SEQ 494ID:138, SEQ ID:139 or SEQ ID:140 SEQ ID:141, SEQ ID: 142, SEQ 531ID:143, SEQ ID:144 or SEQ ID:145 SEQ ID:146, SEQ ID: 147, SEQ 578ID:148, SEQ ID:149 or SEQ ID:150

[0120] Typically, the hybridization methods are conducted understringent conditions. As such stringent conditions, for example, theprehybridization or hybridization treatments are conducted inprehybridization buffer and hybridization buffer, and the washings areconducted twice for 15 minutes in washing buffer. The hybridizationmethods may optionally have another washing for 30 minutes in a buffercontaining 0.1×SSC (0.015M NaCl, 0.0015M sodium citrate) and 0.5% SDS.As the prehybridization buffer, there may be utilized a buffercontaining 6×SSC (0.9M NaCl, 0.09M sodium citrate), 5×Denhart (0.1%(w/v) phycol 400, 0.1% (w/v) polypyrolidone and 0.1% BSA), 0.5% (w/v)SDS and 100 μg/ml of salmon sperm DNA. Also as the prehyblidizationbuffer, there may be utilized a DIG EASY Hyb buffer (Boehringer Manheim)to which salmon sperm DNA is added to a concentration of 100 μg/ml.Further, as the prehybridization buffer, there may be utilized a buffercontaining 6×SSPE (0.9M NaCl, 0.052M NaH₂PO₄, 7.5 mM EDTA), 0.5% SDS,5×Denhart and 0.1 mg/ml of salmon sperm DNA. As the hybridizationbuffer, there may be utilized the prehybridization buffer to which theprobe oligonucleotide is added to a sufficient amount. The temperatureof the prehybridization and hybridization treatments can vary with thelength of the probe oligonucleotide and for example, may be at the Tmvalue of the probe oligonucleotide to a temperature that is 2 to 3 lowerthan the Tm value of the probe oligonucleotide. The temperature of thewashings can also vary with the length of the oligonucleotide, and forexample may be conducted at room temperature. The Tm value in suchcases, can be achieved by estimating the quantity of nucleotide unitsthat should form hydrogen bonds in the hybridization buffer with thenucleotide units in the probe oligonucleotide, and then by adding thetemperatures achieved from adding 2° C. for the A or T nucleotide unitsin the probe oligonucleotide which should form the hydrogen bond andadding 4° C. for the G or T nucleotide units in the probeoligonucleotide which should form the hydrogen bond.

[0121] For example, the hybridization methods can involve dot-blothybridization methods, mismatch detection methods or the like.

[0122] The dot-blot hybridization methods typically involve fixing thetest ERα polynucleotide to a membrane and evaluating whether the probeoligonucleotide can hybridize to the searching region in the fixed testERα polynucleotide. In fixing test ERα polyntucleotide onto themembrane, there can be utilized as the test ERα polynucleotide, thesearching region polynucleotide, the prepared test cDNA, the preparedtest genomic DNA, a purified test ERα polynucleotide or the like. Thetest ERα polynucleotide can be fixed to the membrane by incubating thetest ERα polynucleotide at 90 to 100° C. for 3 to 5 minn, by spottingthe test ERα polynucleotide onto the membrane, by drying the resultingmembrane and by exposing the spotted searching region with UV light. Asthe membrane, there can be utilized a nylon membrane such as Hybond N(Amerscham Pharmacia). The probe oligonucleotide can then be utilizedto, evaluate whether the probe oligonucleotide can hybridize to thesearching region. The probe oligonucleotide may be utilized byincubating the probe oligonucleotide and the test ERα polynucleotide at40 to 50° C. for 10 to 20 hours. The resulting membrane may then bewashed and the hybridized probe oligonucleotide can then be detected, ifpresent.

[0123] When the probe oligonucleotide is radiolabeled with ³²P, thehybridized probe oligonucleotide, if present, may be detected byexposing the resulting membrane to a X-ray film.

[0124] When the probe oligonucleotide is nonradioactively labeled withbiotin, the hybridized probe oligonucleotide, if present, may bedetected with a spacer and a hybridization detection enzyme such asbiotinylated alkaline phosphatase, biotinylated peroxidase or the like.When the probe oligonucleotide labeled with biotin can hybridize to thesearching region, the spacer, such as streptavidin, can bind to thehybridized probe oligonucleotide labeled with biotin such that thehybridization detection enzyme can then connect to the hybridized probeoligonucleotide labeled with biotin through the spacer. The connectedhybridization detection enzyme can then participate in a reaction toindicate whether the probe oligonucleotide has hybridized to thesearching region in the test ERα polynucleotide. The enzymatic reactioncan provide a change in color or a luminescence.

[0125] When the probe oligonucleotide does not hybridize to thesearching region, it can be determined that the searching regioncontains one or more of the variant codons. The searching region maythen be sequenced. The mutation in the variant codon, if present, may bedetermined similarly to the methods described above in the PCRamplification and nucleotide sequencing methods.

[0126] The mismatch detection methods are described in Biswas, I. andHsieh, P., J. Biol. Chem., 271(9), pp. 5040-5048 (1996) as well asNippon gene information, 1999, No. 125, Nippon Gene, Toyama. In suchmismatch detection methods, a mismatch detection enzyme, such as Taq MutS, is utilized to search certain mismatches in the hybridization of theprobe oligonucleotide to the searching region. The mismatch detectionenzyme allows the mismatches in the hybridization of the probeoligonucleotide with the searching region to be detected at hightemperatures such as at a temperature of 75° C. or lower. Typically,such mismatches in the hybridization thereof, bound with the mismatchdetection enzyme, can be detected by a gel shift assay or by the dotblot hybridization methods as described in the above. When the mismatchdetection enzyme can bind to a mismatched hybridization of the probeoligonucleotide and the searching region, it may be determined that thesearching region contains one or more of the valiant codons. Thesearching region may be sequenced. The mutation in the variant codon, ifpresent, may then be determined similarly to the methods described abovein the PCR amplification and nucleotide sequencing methods.

[0127] Further, in the RFLP methods, a restriction enzyme is mixed withthe test ERα polynucleotide under reacting conditions. Typically, therestriction enzyme has a restriction site overlapping with the codon inthe searching region, which is suspected to be the variant codon. Asuccessful or an unsuccessful restriction digest at the restriction sitecan determine whether there is the variant codon in the searchingregion. The results of the restriction digestion can be evaluated by gelelectrophoresis analysis, such as with low melting point agarose gelelectrophoresis. The searching region may then be sequenced, if needed.The mutation in the variant codon, if present, may then be determinedsimilarly to the methods described above in the PCR amplification andnucleotide sequencing methods.

[0128] The phenotype diagnosis methods may involve searching in an aminoacid sequence of the test ERA for one or more substituted amino acidswhich confer the activity for transactivation of the reporter gene asdescribed in the above 5.2. After searching for the substituted aminoacid, the mutation in the test ERα, if present, is determined bycomparing the amino acid sequence of the test ERα to the amino acidsequence of the normal ERα. To search for the substituted amino acid inthe test ERα, the searching region in the test ERα may include the aminoacids in the test ERα at relative positions 303 to 578. For example,such phenotype diagnosis methods may have the searching region includean amino acid in the test ERα at one or more relative positions selectedfrom 303, 309, 390, 396, 415, 494, 531, 578 and the like.

[0129] To search for the substituted amino acid in the test ERα, anantibody having an epitope in the searching region in the test ERα maybe useful. A successful or unsuccessful binding of such an antibody candetermine whether there is a substituted amino acid at the searchingregion in the test ERα. The mutation in the test ERα can then bedetermined by comparing the amino acid sequence of the test ERα with theamino acid sequence of a normal ERα.

[0130] The test ERα may be prepared from a test sample by cell extracttechniques. Further, the test ERα may be prepared for the phenotypediagnosis methods by purifying recombinant test ERα.

[0131] 5.4. The Reporter Assay With the Test ERα

[0132] A test ERα can be assayed for the activity for transactivation ofthe reporter gene, described in the above 5.2., by utilizing an assaycell comprising the test ERα and a chromosome which comprises thereporter gene. In such cases, the assay cell is typically exposed to aligand and the transactivation level of the reporter gene is measured toquantitatively analyze the activity for transactivation of the reportergene by the test ERα. Further, the activity for transactivation of thereporter gene by the test ERα can be evaluated by comparing thetransactivation level of the reporter gene by the test ERα to thetransactivation level of the reporter gene by a standard. Furthermore,the test ERα can be screened by selecting the test ERα in which thetransactivation level of the reporter gene by the test ERα is differentthan the transactivation level by the standard.

[0133] The assay cell can be produced by introducing the reporter geneand a gene encoding the test ERα into a host cell. The reporter gene isinserted into a chromosome of the host cell. The test ERα gene can beintroduced into the host cell for transient expression or can beintroduced into the host cell so that the test ERα gene is inserted intoa chromosome of the host cell. When inserting the test ERα gene into achromosome of the host cell, the test ERα gene may be inserted into thechromosome together with the test ERα or into another chromosome in thehost cell. Additionally, the reporter gene may be introduced into thehost cell to produce a stably transformed cassette cell, as described inthe above 5.2., and the test ERα gene may then be introduced into thestably transformed cassette cell, as described in the above 5.2.

[0134] The test ERα gene is introduced into the host cell so that thetest ERα gene can be expressed in the assay cell to provide the testERα. In this regard, such a test ERα gene typically comprises a promoterlinked operably upstream from a polynucleotide which encodes the testERα.

[0135] To introduce the test ERα gene into the host cell, conventionaltechniques for introducing the test ERα gene may be applied according tothe type of host cell, as described in the above 5.2. In this regard,when test ERα is introduced into the host cell for transient expression,the test ERα gene is introduced in a circular form. When inserting thetest ERα gene into the chromosome of the host cell, the test ERα isintroduced in a linearized form. Also, a vector may be utilized tointroduce the test ERα gene or the reporter gene into the host cell, asdescribed in the above 5.2.

[0136] Further, the test ERα gene can be introduced into the stablytransformed cassette cell to provide the assay cell. In such cases, thetest ERα gene may also be introduced into the stably transformedcassette cell to provide a stably transformed binary cell. Such anstably transformed binary cell has the chromosomes thereof stablycomprise the test ERα gene and the reporter gene.

[0137] The host cell utilized to produce the assay cell typically failshave an expressed normal or mutant ERα. Examples of the host cellsinclude eukaryotic cells such as HeLa cells, CV-1 cells, Hepa1 cells,NIH3T3 cells, HepG2 cells, COS1 cells, BF-2 cells, CHH-1 cells and thelike.

[0138] In the reporter assay, the assay cell is typically exposed with asufficient amount of a ligand for one to several days. Further, theligand can be exposed to the assay cell under agonistic conditions orantagonistic conditions directed to the test ERα. The agonisticconditions typically have the assay cell exposed to the ligand as thesole agent probable of stimulating the test ERα. The antagonisticconditions typically have the assay cell exposed to the ligand and E2.

[0139] As the ligand there is usually utilized a ligand that is purelyor partially antagonistic or agonistic to the normal ERα. Examples ofsuch ligands include the partial anti-estrogens such as tamoxifen,4-hydroxytamoxifen and raloxifene, the pure anti-estrogens such as ICI182780 (Wakeling A E et al., Cancer Res., 512:3867-3873 (I991)) and ZM189154 (Dukes M et al., J. Endocrinol., 141:335-341 (1994)) and thelike.

[0140] After exposure, the transactivation level of the reporter gene ismeasured by measuring the expression level of the reporter gene. In suchcases, the reporter protein or the reporter RNA (encoded by the reportersequence) is stored in the cell or is secreted from the cell so that theexpression level can be measured therewith. The expression level of thereporter gene can be measured by a Northern blot analysis, by a Westernblot analysis or by measuring the activity level of the reporterprotein. The activity level of the protein typically indicates the levelat which the reporter protein is expressed.

[0141] For example, when the reporter gene encodes luciferase as thereporter protein, the expression level of the reporter gene can bemeasured by the luminescence provided by reacting luciferin andluciferase. In such cases, a crude cell extract is produced from thecells and luciferin is added to the crude cell extract. The luciferinmay be allowed to react with the luciferase in the cell extract at roomtemperature. The luminescence from adding luciferin is usually measuredas an indicator of the expression level of the reporter gene, since thecrude cell extract produces a luminescence at a strength proportional tothe level of luciferase expressed in the cell and present in the crudecell extract. A luminometer may be utilized to measure the luminescencein the resulting crude cell extract.

[0142] The measured transactivation level can then be compared with atransactivation level of the reporter gene by a standard to evaluate theactivity for transactivation by the test ERα. Such a transactivationlevel of the reporter gene by the standard in evaluating the activityfor transactivation by the test ERα can be the expected transactivationlevel of the reporter gene in cases in which the assay cell expressesthe normal ERα or an ERα which phenotype is known (instead of the testERα). When the measured transactivation level provided by the test ERαis different than the transactivation level of the reporter gene by thestandard, the test ERα may be selected as a mutant ERα.

[0143] Furthermore, mutant ligand dependent transcription factors can bescreened. In such cases, the a gene encoding the test ligand dependenttranscription factor, instead of the test ERα gene, in introduced intothe host cell. Examples of such test ligand dependent transcriptionfactors include a test ERβ, a test AR, a test GR, a test TR, a test PR,a test PXR, a test lipophilic vitamin receptor such a test VDR and atest RAR and the like. The reporter gene in such cases comprises anappropriate receptor responsive sequence cognate with the provided testligand dependent transcriptional factor, instead of the ERE.

6. EXAMPLES 6.1. Example 1 A Polynucleotide Encoding the Human MutantERα

[0144] 6.1.1. Production of a Plasmid Encoding Human Normal ERα

[0145] A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1)is utilized to specifically PCR amplify therefrom a cDNA encoding ahuman normal ERα. The PCR mixture in this PCR amplification contains 10ng of the human liver cDNA library, 10 pmol of an oligonucleotidedepicted in SEQ ID: 11, 10 pmol of a oligonucleotide depicted in SEQ ID:12, LA-Taq Polymerase (Takara Shuzo), the buffer provided with theLA-Taq Polymerase and dNTPs (dATP, dTTP, dGTP, dCTP). Theoligonucleotides depicted in SEQ ID: 11 and SEQ ID: 12 are synthesizedwith a DNA synthesizer (Model 394, Applied Biosystems). In this PCRamplification, there is repeated 35 times with a PCRsystem 9700 (AppliedBiosystems), an incubation cycle entailing an incubation at 95° C. for 1minute followed by an incubation at 68° C. for 3 minutes.

[0146] The resulting PCR mixture is subjected to low melting pointagarose gel electrophoresis (Agarose L: Nippon Gene) to confirm that theamplified cDNA from the PCR amplification has a size of about 1.8 kb.After recovering the amplified cDNA from the low melting point agarosegel, a sample of the recovered cDNA is prepared with a Dye TerminatorSequence Kit FS (Applied Biosystems). The prepared sample of the cDNA issequenced with an ABI autosequencer (Model 377, Applied Biosystems), toreveal that the cDNA has a nucleotide sequence encoding a human normalERα which has the amino acid sequence shown in SEQ ID: 1.

[0147] Another PCR amplification is then similarly conducted to add aKozak consensus sequence immediately upstream from the start codon (ATC)in the cDNA. In this PCR amplification, there is utilized 100 ng of thecDNA, a oligonucleotide depicted in SEQ ID: 151 and an oligonucleotidedepicted in SEQ ID: 12. The resulting PCR mixture is subjected to lowmelting point agarose gel electrophoresis (Agarose L: Nippon Gene) toconfirm that the amplified cDNA from the PCR amplification has a size ofabout 1.8 kb. After recovering the amplified cDNA from the low meltingpoint agarose gel, 1 μg of the amplified cDNA is treated with a DNABlunting Kit (Takara Shuzo) to blunt the ends of the amplified cDNA.Subsequently, the resulting cDNA therefrom is allowed to react with a T4polynucleotide kinase to phosphorylate the ends thereof. After phenoltreating the phosphorylated cDNA, the phosphorylated cDNA is ethanolprecipitated to achieve a purified form of the phosphoylated cDNA.

[0148] The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with bacterial alkalinephosphatase (BAP) for 1 hour at 65° C. The restriction digested pRc/RSVis then purified by a phenol treatment and ethanol precipitation. Therestriction digested pRc/RSV is treated with a DNA Blunting Kit (TakaraShuzo) to blunt the ends thereof and is subjected to low melting pointagarose gel electrophoresis (Agarose L, Nippon Gene). After recoveringthe restriction digested pRc/RSV from the low melting point agarose gel,100 ng of the restriction digested pRc/RSV and all of the above purifiedform of the phosphorylated cDNA are used in a ligation reaction with aT4 DNA ligase. The ligation reaction mixture is used to transform E.coli competent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB (Luria-Bertani) medium to which ampicillin is added to aconcentration of 50 μg/ml (hereinafter referred to as LB-amp medium; J.Sambrook, E. F. Frisch, T. Maniatis; Molecular Cloning 2nd Edition, ColdSprings Harbor Laboratory Publishing, 1989). The clones thereof showingan ampicillin resistance are then recovered. Some of the clones are thenused to isolate therefrom the plasmids derived from the ligationreaction. An aliquot sample of each of the isolated plasmids are thenprepared with a Dye Terminator Sequence Kit FS (Applied Biosystems). Theprepared plasmids are sequenced with an ABI autosequencer (Model 377,Applied Biosystems), to confirm that there is a plasmid that has anucleotide sequence encoding human normal ERα having the amino acidsequence shown in SEQ ID: 1. Such a plasmid is selected and isdesignated as pRc/RSV-hERαKozak.

[0149] 6.1.2. Production of Plasmids Encoding the Human Mutant ERαK303R,S309F, M396V, G415V, G494V or K531E

[0150] 6.1.2.1. Production of a Plasmid for Mutagenisis

[0151] The plasmid pRc/RSV-hERαKozak is restriction digested withrestriction enzyme Not I for 1 hour at 37° C. The restriction digestionreaction mixture is subjected to low melting point agarose gelelectrophoresis (Agarose L: Nippon Gene) to confirm that there is DNAfragment having a size of about 1.6 kb. The 1.6 kb DNA fragment is thenrecovered from the low melting point agarose gel.

[0152] The plasmid pBluescriptII SK(+) (Stratagene) is restrictiondigested with NotI for 1 hour at 37° C. and is then treated with BAP for1 hour at 65° C. The restriction digestion reaction mixture is subjectedto low melting point agarose gel electrophoresis (Agarose L: NipponGene) and the restriction digested pBluescriptII SK(+) is recovered fromthe low melting point agarose gel. Subsequently, 100 ng of the above 1.6kb DNA, fragment and 100 ng of the recovered pBluescriptII SK(+) areused in a ligation reaction with T4 DNA ligase. The ligation reactionmixture is used to transform E. coli competent DH5α cells (TOYOBO). Thetransformed E. coli cells are cultured in LB-amp medium. The clonesthereof showing an ampicillin resistance are then recovered. Some of theclones are then used to isolate therefrom the plasmids derived from theligation reaction. An aliquot sample of each of the isolated plasmidsare then restriction digested with restriction enzymes Not I and HindIII. The restriction digestion reaction mixtures are subjected toagarose gel electrophoresis. It is then confirmed that there is plasmidin which the plus strand in the plasmid contains the sense strandencoding the human normal ERα operably with M13 microphage replicationorigin (fl ori). In this regard, it is confirmed that there is a plasmidwhich has a structure such that when the fl ori replicates one of thestrands in the plasmid, the sense strand encoding human normal ERα wouldbe replicated therewith. Such a plasmid is selected and is designated aspSK-NN.

[0153] 6.1.2.2. Site Directed Mutagenesis at Relative Positions 303,309, 396, 415, 494 and 531

[0154] According to the methods described in McClary J A et al.(Biotechniques 1989(3): 282-289), specified mutations are introducedinto the polynucleotide encoding the human normal ERα. Such proceduresare described in relation with the present invention below.

[0155] The plasmid pSK-NN, provided in the above 6.1.2.1., is utilizedto transform E. coli competent CJ236 cells (Takara Shuzo) according tothe protocol provided with the E. coli competent CJ236 cells. A clonethereof showing ampicillin resistance is then cultured for 16 hours in aLB-amp medium. Subsequently, a colony of the clone is suspended in 10 mlof a 2×YT medium to which a M13 helper phage is added to a concentrationof at least 1×10¹¹pfu/ml (hereinafter referred to as 2×YT-M13) medium.After culturing the clone in the 2×YT-M13 medium for 2 hours at 37° C.,kanamycin is added thereto to a concentration of 50 μg/ml and the cloneis then cultured for 22 hours. The resulting suspension is centrifugedand 8 ml of the resulting supernatant is transferred to a 15 ml testtube. Two milliliters (2 ml) of 2.5M NaCl-40% PEG8000 (Sigma) is thenadded to and stirred with the supernatant. The supernatant isrefrigerated at 4° C. for 1 hour and is centrifuged (3,000 rpm, 2,000×g,10 minutes, 4° C.) to collect the phage therefrom as a pellet. After thephage is suspended in 400 μl of distilled water, an identical amount byvolume of phenol is added thereto and the resulting suspension is gentlyshook for 5 minutes. The resulting suspension is centrifuged so that theaqueous layer therein is extracted therefrom. For a second round ofphenol treatment, an identical amount by volume of phenol is then addedto the aqueous layer and is vigorously shook. The resulting suspensionis centrifuged so that the aqueous layer is extracted therefrom. To theaqueous layer from the second phenol treatment, an identical amount byvolume of chloroform is added thereto and is vigorously shook. Theresulting suspension is centrifuged (15,000 rpm, 20,000×g, 5 minutes, 4°C.) to extract the aqueous layer therefrom. To the aqueous layer fromthe chloroform treatment, there is added 800 μl of 100% ethanol and 50μlof 3M sodium acetate. After refrigerating the aqueous layer therefromat −80° C. for 20 minutes, the aqueous layer is centrifuged. Theresulting pellet therefrom is rinsed with 70% ethanol and is then dried.After pellet the residue in sterile water, the light absorbance ofaqueous solution is measured at a wavelength of 260 nm to calculate theamount of the single strand sense DNA encoding human normal ERα therein.

[0156] The oligonucleotides for the site directed mutagenesis aresynthesized to provide the oligonucleotides depicted in SEQ ID: 152, SEQID: 153, SEQ ID: 154, SEQ ID: 155, SEQ ID: 156 and SEQ ID: 157.

[0157] In using the oligonucleotide depicted in SEQ ID: 152, the AAGcodon encoding the lysine present at relative position 303 is changed toan AGG codon encoding arginine.

[0158] In using the oligonucleotide depicted in SEQ ID: 153, the TCCcodon encoding the serine present at relative position 309 is changed toa TTC codon encoding phenylalanine.

[0159] In using the oligonucleotide depicted in SEQ ID: 154, the ATGcodon encoding the methionine present at relative position 396 ischanged to an GTG codon encoding valine.

[0160] In using the oligonucleotide depicted in SEQ D: 155, the GGAcodon encoding the glycine present at relative position 415 is changedto a GTA codon encoding valine.

[0161] In using the oligonucleotide depicted in SEQ ID: 156, the GGCcodon encoding the glycine present at relative position 494 is changedto a GTC codon encoding valine.

[0162] In using the oligonucleotide depicted in SEQ ID: 157, the AAGcodon encoding the lysine present at relative position 531 is changed toa GAG codon encoding glutamic acid.

[0163] Each of the oligonucleotides is phosphorylated with 10 pmol of apolynucleotide kinase (Takara Shuzo) in the buffer provided with thepolynucleotide kinase. In the phosphorylation reactions, 2 mM of ATP isused in each of the reaction mixtures and the reaction mixtures areincubated at 37° C. for 30 minutes. Subsequently, about 1 pmol of thephosphoylated oligonucleotides are mixed, respectively, with 0.2 pmol ofthe single stand sense DNA encoding normal ERα. To produce 10 μlannealing reaction mixtures, the mixtures are then added, respectively,to annealing buffer (20 mM of Tris-Cl(pH7.4), 2 mM of MgCl₂, 50mM ofNaCl). The annealing reaction mixtures are subjected to an incubation at70° C. for 10 minutes, then an incubation at 37° C. for 60 minutes,which is followed by an incubation at 4° C. Synthesizing reactionmixtures are then produced therefrom by adding, respectively, to theannealing reaction mixtures, 2 units (0.25 μl) of T7 DNA polymerase (NewEngland Labs), 2 units of (0.25 μl) of T4 DNA ligase (Takara Shuzo) and1.2 μl of a synthesizing buffer (175 mM of (Tris-Cl (pH 7.4), 375 nM ofMgCl₂, 5 mM of DTT, 4 mM of dATP, 4 mM of dCTP, 4 mM of dGTP, 4mM ofdTTP and 7.5 mM of ATP). The synthesizing reaction mixtures areincubated at 4° C. for 5 minutes, incubated at room temperature for 5minutes, and then incubated at 37° C. for 2 hours, to providesynthesized DNA plasmids.

[0164] Two microliters (2 μl) of each of the synthesizing reactionmixtures are then used to transform E. coli competent DH5α cells(TOYOBO). The transformed E. coli cells are cultured in LB-amp. Theclones thereof showing an ampicillin resistance are then recovered. Someof the clones are then used to isolate therefrom the plasmids from thesynthesizing reactions. An aliquot sample of each of the isolatedplasmids are then prepared with a Dye Terminator Sequence Kit FS(Applied Biosystems). The isolated plasmids are sequenced with an ABIautosequencer (Model 377, Applied Biosystems).

[0165] It is confirmed from the sequencing that the isolated plasmidssynthesized from utilizing the oligonucleotide depicted in SEQ ID: 152provides an isolated plasmid which has in the nucleotide sequenceencoding the human mutant ERα, an AGG codon corresponding to relativeposition 303, to provide arginine. Such an isolated plasmid is selectedand is designated as pSK-NN303.

[0166] It is confirmed from the sequencing that the isolated plasmidssynthesized from the oligonucleotide depicted in SEQ ID: 153 provides anisolated plasmid which has in the nucleotide sequence encoding the humanmutant ERα, a TTC codon corresponding to relative position 309, toprovide phenylalanine. Such an isolated plasmid is selected and isdesignated as pSK-NN309.

[0167] It is confirmed from the sequencing that the isolated plasmidssynthesized from the oligonucleotide depicted in SEQ ID: 154 provides anisolated plasmid which has in the nucleotide sequence encoding the humanmutant ERα, a GTG codon corresponding to relative position 396, toprovide valine. Such an isolated plasmid is selected and is designatedas pSK-NN396.

[0168] It is confirmed from the sequencing that the isolated plasmidssynthesized from the oligonucleotide depicted in SEQ ID: 155 provides anisolated plasmid which has in the nucleotide sequence encoding the humanmutant ERα, a GTA codon corresponding to relative position 415, toprovide valine. Such an isolated plasmid is selected and is designatedas pSK-NN415.

[0169] It is confirmed from the sequencing that the isolated plasmidssynthesized from the oligonucleotide depicted in SEQ ID: 156 provides anisolated plasmid which has in the nucleotide sequence encoding the humanmutant ERα, a GTC codon corresponding to relative position 494, toprovide valine. Such an isolated plasmid is selected and is designatedas pSK-NN494.

[0170] It is confirmed from the sequencing that the isolated plasmidssynthesized from the oligonucleotide depicted in SEQ ID: 157 provides anisolated plasmid which has in the nucleotide sequence encoding the humanmutant ERα, a GAG codon corresponding to relative position 531, toprovide glutamic acid. Such as isolated plasmid is selected and isdesignated as pSK-NN531.

[0171] Table 4 below shows the utilized oligonucleotide for themutagenesis, the produced plasmid therefrom and the resulting humanmutant ERα therefrom. TABLE 1 SEQ ID of utilized oligonucleotideproduced plasmid human mutant ERα SEQ ID:152 pSK-NN303 human mutantERαK303R SEQ ID:153 pSK-NN309 human mutant ERαS309F SEQ ID:154 pSK-NN396human mutant ERαM396V SEQ ID:155 pSK-NN415 human mutant ERαG415V SEQID:156 pSK-NN494 human mutant ERαG494V SEQ ID:157 pSK-NN531 human mutantERαK531E

[0172] The plasmids pSK-NN303, pSK-NN309, pSK-NN396, pSK-NN415,pSK-NN494 and pSK-NN531 are each restriction digested with restrictionenzyme Not I at 37° C. for 1 hour. Each of the restriction digestionreaction mixtures are then subjected to low melting point agarose gelelectrophoresis to confirm that there are DNA fragments having a size ofabout 1.6 kb. The 1.6 kb DNA fragments are then recovered from the lowmelting point agarose gel.

[0173] The plasmid pRc/RSV-hERαKozak, provided in 6.1.1., is restrictiondigested with restriction enzyme Not I at 37° C. for 1 hour and istreated with BAP at 65° C. for 1 hour. The restriction digestionreaction mixture is then subjected to low melting point agarose gelelectrophoresis to confirm that there is a DNA fragment having a size ofabout 5.5 kb. The 5.5 kb DNA fragment is then recovered from the lowmelting point agarose gel.

[0174] Subsequently, 100 ng of the recovered 5.5 kb DNA fragments aremixed, respectively, with 100 ng of the above 1.6 kb DNA fragments for aligation reaction with T4 DNA ligase. The ligation reaction mixtures areused to transform E. coli competent DH5α cells (TOYOBO). The transformedE. coli cells are cultured in LB-amp medium. The clones thereof showingan ampicillin resistance are then recovered. Some of the clones are thenused to isolate therefrom the plasmids derived from the ligationreactions. An aliquot sample of each of the isolated plasmids are thenrestriction digested with either restriction enzyme Not I or Mlu I. Therestriction digestion reaction mixtures are then subjected to agarosegel electrophoresis. It is confirmed that there are isolated plasmidswhich result from the each of the restriction digestions, DNA fragmentshaving the desired sizes. Such isolated plasmids provide DNA fragmentshaving sizes of 5.5 and 1.6 kb in the restriction digestions withrestriction enzyme Not I and provide DNA fragments of 7.1 kb inrestriction digestions with restriction enzyme Mlu I.

[0175] Each of the plasmids above is then PCR amplified witholigonucleotides depicted in SEQ ID: 158, SEQ ID: 159 and SEQ ID: 160.The PCR mixtures in these PCR amplifications contain one of theplasmids, the oligonucleotide depicted in SEQ ID: 158, theoligonucleotide depicted in SEQ ID: 159, the oligonucleotide depicted inSEQ ID: 160, 400 μM of dNTPs (100 μM of dATP, 100 μM of dTTP, 100 μM ofdGTP and 100 μM of dCTP), recombinant Taq DNA polymerase (Takara Shuzo),the PCR buffer provided with the recombinant Taq DNA polymerase. Inthese PCR amplifications, there are repeated 30 times, an incubationcycle entailing an incubation at 94° C. for 30 seconds, then anincubation at 65° C. for 1 minute, which is followed by an incubation at72° C. for 1 minute and 45 seconds. Ten microliters (10 μl) of each ofthe resulting 25 μl PCR mixtures are subjected to a 1% agarose gelelectrophoresis (Agarose S, Nippon Gene) to confirm that the resultingplasmids have a size of about 1.2 kb. The plasmids are then preparedwith a Dye Terminator Sequence Kit FS (Applied Biosystems). The preparedsamples of the plasmids are sequenced, respectively, with an ABIautosequencer (Model 377, Applied Biosystems).

[0176] It is confirmed from the sequencing that the plasmid derived frompSK-NN303 encodes the human mutant ERαK303R (AAG→AGG; lysine→arginine;relative position 303). This plasmid is designated as pRc/RSV-hERαK303RKozak.

[0177] It is confirmed from the sequencing that the plasmid derived frompSK-NN309 encodes the human mutant ERαS309F (TCC→TTC;serine→phenylalanine; relative position 309). This plasmid is designatedas pRc/RSV-hERαS309F Kozak.

[0178] It is confirmed from the sequencing that the plasmid derived frompSK-NN396 encodes the human mutant ERαM396V (ATG→GTG; methionine→valine;relative position 396). This plasmid is designated as pRc/RSV-hERαM396VKozak.

[0179] It is confirmed from the sequencing that the plasmid derived frompSK-NN415 encodes the human mutant ERαG415V (GGA→GTA; glycine→valine;relative position 415). This plasmid is designated as pRc/RSV-hERαG415VKozak.

[0180] It is confirmed from the sequencing that the plasmid derived frompSK-NN494 encodes the human mutant ERαG494V (GGC→GTC; glycine→valine;relative position 494). This plasmid is designated as pRc/RSV-hERαG494VKozak.

[0181] It is confirmed from the sequencing that the plasmid derived frompSK-NN531 encodes the human mutant ERαK531E (AAG→GAG; lysine→glutamicacid; relative position 531). This plasmid is designated aspRc/RSV-hERαK531E Kozak.

[0182] Table 5 below shows the plasmid utilized to produce the plasmidand the resulting plasmid produced therefrom. TABLE 5 encoded plasmidproduced plasmid human mutant ERα pSK-NN303 pRc/RSV-hERK303R Kozak humanmutant ERαK303R pSK-NN309 pRc/RSV-hERS309F Kozak human mutant ERαS309FpSK-NN396 pRc/RSV-hERM396V Kozak human mutant ERαM396V pSK-NN415pRc/RSV-hERG415V Kozak human mutant ERαG415V pSK-NN494 pRc/RSV-hERG494VKozak human mutant ERαG494V pSK-NN531 pRc/RSV-hERK531E Kozak humanmutant ERαK531E

[0183] 6.1.3. Production of Plasmids Encoding the Human Mutant ERαG390D,S578P or G390D/S578P

[0184] 6.1.3.1. Production of Plasmids Encoding the Human MutantERαG390D and S578P

[0185] The QuickChange Site-Directed Mutagenesis Kit (Stratagene) isused to mutagenize the plasmid pRc/RSV-hERαKozak, described in the above6.1.1., so that the mutagenized plasmid encodes the human mutantERαG390D or the human mutant ERαS578P. In using the oligonucleotidesdepicted in SEQ ID: 17 and SEQ ID: 18, the GGT codon encoding theglycine present at relative position 390 is changed to a GAT variantcodon encoding aspartic acid. In using the oligonucleotides depicted inSEQ ID: 27 and SEQ ID: 28, the TCC codon encoding the serine present atrelative position 578 is changed to a CCC variant codon encodingproline. The manual provided with the QuickChange Site-DirectedMutagenesis Kit is used to produce the plasmids pRc/RSV-hERαG390D Kozak(GGT→GAT; glycine→asapartic acid; relative position 390) andpRc/RSV-hERαS578P Kozak (TCC→CCC; serine→proline; relative position578). The plasmids pRc/RSV-hERαG390D Kozak and pRc/RSV-hERαS578P Kozakare sequenced to confirm that the plasmids encoding the human mutant ERαcontain the desired mutation therein at relative position 390 or 578.

[0186] The QuickChange Site directed Mutagenesis Kit (Stratagene) isthen used to mutagenize pRc/RSV-hERαG390D Kozak so that the mutagenizedplasmid encodes the human mutant ERαG390D/S578P. The oligonucleotidesdepicted in SEQ ID: 27 and SEQ ID: 28 are used to produce plasmidpRc/RSV-hERαG390D/S578P Kozak (GGT→GAT; glycine→asapartic acid; relativeposition 390 and TCC→CCC; serine→proline; relative position 578). Theplasmid pRc/RSV-hERαG390D/S578P Kozak is sequenced to confirm that theplasmid encoding the human mutant ERα contains the desired mutationstherein at relative positions 390 and 578.

[0187] 6.1.3.2. Preparation From a Test Human Liver Tissue Sample of aPlasmid Encoding a Human Mutant ERαG390D/S578P

[0188] A frozen sample of test human liver tissue was utilized to obtaina polynucleotide encoding a human mutant ERαG390D/S578P. In utilizingthe test human liver tissue sample, 0.1 g of the test human liver tissuesample was homogenized with a homogenizer in 5 ml of a buffer containing4M guanidium thiocyanate, 0.1M Tris-HCl (pH 7.5) and 1% βmercaptoethanol. The resulting buffer was layered with 25 ml of anaqueous 5.7M CsCl solution and was ultracentrifuged at 90,000×g for 24hours to obtain a RNA pellet. After rinsing the RNA pellet with 70%ethanol, the RNA pellet was allowed to dry at room temperature. The RNApellet was then dissolved in 10 μl of sterile water to a concentrationof 1.2 μg/ml. Test cDNAs were then produced by collectively using theRNAs in the RNA solution as a template in a reverse transcriptionreaction. In producing the test cDNAs, reverse transcriptase(Superscript II; GibcoBRL) was used with 1 μl of the RNA solution,oligo-dT oligonucleotides (Amerscham Pharmacia) and the buffer providedwith the reverse transcriptase. The reverse transcription reaction wasallowed to react for 1 hour at 37° C., to provide the above the testcDNAs.

[0189] Similarly to the above 6.1.1., 1/50 by volume of the test cDNAswere used to produce pRc/RSV-hERαG390D/S578P Kozak. In this regard, thetest cDNAs were used to specifically PCR amplify therefrom witholigonucleotides depicted in SEQ ID: 11 and SEQ ID: 12, the cDNAencoding the human mutant ERαG390D/S578P. The cDNA encoding the humanmutant ERαG390D/S578P was then PCR amplified with the oligonucleotidesdepicted in SEQ ID: 151 and SEQ ID: 12 to add a Kozak consensus sequenceimmediately upstream from the start codon (ATG) in the cDNA. Theamplified product was then inserted into the HindIII site of the plasmidpRc/RSV to provide pRc/RSV -hERαG390D/S578P Kozak.

6.2. Example 2 Production of a Plasmid Containing the Reporter Gene

[0190] An oligonucleotide depicted in SEQ ID: 161 and an oligonucleotidehaving a nucleotide sequence complementary thereto were synthesized witha DNA synthesizer. The oligonucleotide depicted in SEQ ID: 161 wassynthesized to encode one of the strands of an ERE derived from theupstream region in a Xenopus vitellogenin gene. The secondoligonucleotide was synthesized to have a nucleotide sequencecomplementary to the oligonucleotide depicted in SEQ ID: 161. The twooligonucleotides were annealed together to produce a DNA encoding an ERE(hereinafter referred to as the ERE DNA). The ERE DNA was then ligatedtogether with a T4 DNA ligase to provide a EREx5 DNA having a 5 tandemrepeat of the ERE. A T4 polynucleotide kinase was allowed to react withthe EREx5 DNA to phosphorylate the ends thereof.

[0191] An oligonucleotide depicted in SEQ ID: 162 and an oligonucleotidedepicted in SEQ ID: 163 were then synthesized with a DNA synthesizer.The oligonucleotide depicted in SEQ ID: 162 was synthesized to encodeone of the strands in the nucleotide sequence of a TATA sequence derivedfrom the mouse metallothionein I gene and the leader sequence thereof.The oligonucleotide depicted in SEQ ID: 163 was synthesized to encode anucleotide sequence complementary to the oligonucleotide depicted in SEQID: 162. The oligonucleotides depicted in SEQ ID: 162 and SEQ ID: 163were annealed together to produce a DNA encoding the TATA sequence. A T4polynucleotide kinase was allowed to react with the DNA encoding theTATA sequence to phosphorylate the ends thereof.

[0192] The plasmid pGL3 (Promega), which encodes the firefly luciferasegene, was restriction digested with restriction enzymes Bgl II and HindIII and was then treated with BAP at 65° C. for 1 hour. The restrictiondigestion reaction mixture was then subjected to low melting pointagarose gel electrophoresis (Agarose L, Nippon Gene) to confirm thatthere was a DNA fragment having the nucleotide sequence encoding thefirefly luciferase. The DNA fragment having the nucleotide sequenceencoding the firefly luciferase was then recovered from the low meltingpoint agarose gel. Subsequently, 100 ng of the recovered DNA fragmentand 1 μg of the DNA encoding the TATA sequence were used in a ligationreaction with T4 DNA ligase to provide a plasmid pGL3-TATA.

[0193] The plasmid pGL3-TATA was restriction digested with restrictionenzyme Sma I and was then treated with BAP at 65° C. for 1 hour. Therestriction digestion reaction mixture was then subjected to low meltingpoint agarose gel electrophoresis (Agarose L, Nippon Gene) to confirmthat there was a DNA fragment encoding the TATA sequence and the fireflyluciferase. After recovering such a DNA fragment from the low meltingpoint agarose gel, 100 ng of the recovered DNA fragment and 1 μg of theEREx5 DNA were used in a ligation reaction with T4 DNA ligase to providea plasmid pGL3-TATA-EREx5.

[0194] The plasmid pUCSV-BSD (Funakoshi) was restriction digested withrestriction enzyme BamH I to prepare a DNA encoding a blasticidin Sdeaminase gene expression cassette. Further, the plasmid pGL3-TATA-EREx5was restriction digested with restriction enzyme BamH I and was thentreated with BAP at 65° C. for 1 hour. The DNA fragment encoding ablasticidin S deaminase gene expression cassette was then mixed with therestriction digested pGL3-TATA-EREx5. The mixture was then used in aligation reaction with T4 DNA ligase to provide plasmids. The ligationreaction mixture was used to transform E. coli competent DH5α cells. Thetransformed cells are cultured in LB-amp. The clones thereof showing anampicillin resistance are then recovered. Some of the clones are thenused to isolate therefrom the plasmids derived from the ligationreaction. An aliquot sample of each of the isolated plasmids are thenrestriction digested with restriction enzyme BamH I. The restrictiondigestion reaction mixtures were then subjected to agarose gelelectrophoresis to confirm whether there was a plasmid which has astructure in which the DNA encoding a blasticidin S deaminase geneexpression cassette has been inserted into the Bam HI restriction sitein pGL3-TATA-EREx5. The plasmid having such a structure was selected andwas designated as pGL3-TATA-EREx5-BSD.

6.3. Example 3 Production of a Stably Transformed Cassette Cell

[0195] In order to produce stably transformed cassette cells, whichstably contain in one of its chromosomes the reporter gene produced in6.2. (hereinafter referred to as the ERE reporter gene), the plasmidpGL3-TATA-EREx5-BSD was linearized and introduced into HeLa cells.

[0196] The plasmid pGL3-TATA-EREx5-BSD was restriction digested withrestriction enzyme Sal I to linearize pGL3-TATA-EREx5-BSD.

[0197] Approximately 5×10⁵ HeLa cells were cultured as host cells for 1day using culture dishes having a diameter of about 10 cm (Falcon) inDMEM medium (Nissui Pharmaceutical Co.) containing 10% FBS at 37° C.under the presence of 5% CO₂.

[0198] The linearized pGL3-TATA-EREx5-BSD were then introduced to thecultured HeLa cells by a lipofection method using lipofectamine (LifeTechnologies). According with the manual provided with thelipofectamine, the conditions under the lipofection method included 5hours of treatment, 7 μg/dish of the plasmids above and 21 μl/dish oflipofectamine.

[0199] After the lipofection treatment, the DMEM medium was exchangedwith DMEM medium containing 10% FBS and the transformed HeLa cells werecultured for about 36 hours. Next, the transformed HeLa cells wereremoved and collected from the dish by trypsin treatment and weretransferred into a container containing a medium to which blasticidin Swas added to a concentration of 16 μg/ml. The transformed HeLa cellswere cultured in such medium containing blasticidin S for 1 month whileexchanging the medium containing blasticidin S every 3 or 4 days to afresh batch of the medium containing blasticidin S.

[0200] The resulting clones, which were able to proliferate and producea colony having a diameter of from 1 to several mm, were transferred asa whole to the wells of a 96-well ViewPlate (Berthold) to which mediumhad previously been dispensed thereto. The colonies of the clones werefurther cultured. When the colonies proliferated to such a degree thatthey covered 50% or more of the bottom surface of the well (about 5 daysafter the transfer), the clones were removed and collected by trypsintreatment. The clones then were divided into 2 subcultures. One of thesubcultures was transferred to a 96-well ViewPlate, which was designatedas the master plate. The other subculture was transferred to a 96-wellViewPlate, which was designated as the assay plate. The master plate andthe assay plate contained medium so that the clones could be cultured.The master plate was continuously cultured under similar conditions.

[0201] After culturing the subcultures in the assay plate for 2 days,the medium was then removed from the wells of the assay plate and theclones attached to the well walls were washed twice with PBS(−). A5-fold diluted lysis buffer PGC50 (Toyo Ink) was added to thesubcultures in the wells of the assay plate at 20 μl per well. The assayplate was left standing at room temperature for 30 minutes and were seton a luminometer LB96P (Berthold), which was equipped with an automaticsubstrate injector. Subsequently, 50 μl of the substrate solution PGL100(Toyo Ink) was automatically dispensed to each of the lysed clones inthe assay plate to measure the luciferase activity therein with theluminometer LB96P. Ten (10) clones, which exhibited a high luciferaseactivity were selected therefrom.

[0202] Samples of the clones in the master plate, which correspond tothe selected 10 clones were then cultured at 37° C. for 1 to 2 weeks inthe presence of 5% CO₂ using dishes having a diameter of about 10 cm(Falcon) in medium.

[0203] The plasmid pRc/RSV-hERαKozak was then introduced to the selectedclones by a lipofection method using lipofectamine (Life Technologies)to provide a second round of clones. According with the manual providedwith the lipofectamine, the conditions under the lipofection methodincluded 5 hours of treatment, 7 μg/dish of the plasmids above and 21μl/dish of lipofectamine. A DMSO solution containing 17β-E2 was thenadded to the resulting second clones to a concentration of 10 nM. Afterculturing the second clones for 2 days, the luciferase activity wasmeasured, similarly to the above, for each of the second clones. Theclone in the master plate, which provided the second clone exhibitingthe highest induction of luciferase activity, was selected as the stablytransformed cassette cell which stably contained in one of itschromosomes the ERE reporter gene (hereinafter referred to as the stablytransformed ERE cassette cell).

6.4. Example 4 Production of Stably Transformed Binary Cells

[0204] Four stably transformed cells containing the ERE reporter genewith the human mutant ERαG390D, S578P or G390D/S578P or human normal ERα(hereinafter referred to as the stably transformed ERE binary cells)were produced. The first stably transformed ERE binary cell contained inits chromosomes the linearized pGL3-TATA-EREx5-BSD, which encodes thereporter gene, and the linearized pRc/RSV-hERαKozak, which encodes thehuman normal ERα. The second stably transformed ERE binary cellcontained in its chromosomes the linearized pGL3-TATA-EREx5-BSD, whichencodes the ERE reporter gene, and the linearized pRc/RSV-hERαG390DKozak, which encodes the human mutant ERαG390D. The third stablytransformed ERE binary cell contained in its chromosomes the linearizedpGL3-TATA-EREx5-BSD, which encodes the ERE reporter gene, and thelinearized pRc/RSV-hERαS578P Kozak, which encodes the human mutantERαS578P. The fourth stably transformed ERE binary cell contained in itschromosomes the linearized pGL3-TATA-EREx5-BSD, which encodes the EREreporter gene, and the linearized pRc/RSV-hERαG390D/S578P Kozak, whichencodes the human mutant ERαG390D/S578P.

[0205] In order to produce the stably transformed ERE binary cells, theplasmids pGL3-TATA-EREx5-BSD, pRc/RSV-hERαG390D Kozak, pRc/RSV-hERαS578PKozak and pRc/RSV-hERαG390D/S578P Kozak were each linearized andintroduced into HeLa cells. To linearize, the plasmids above wererestriction digested with restriction enzyme Sal I.

[0206] Approximately 5×10⁵ HeLa cells were cultured as host cells for 1day using dishes having a diameter of about 10 cm (Falcon) in DMEMmedium (Nissui Pharmaceutical Co.) containing 10% FBS at 37° C. underthe presence of 5% CO₂.

[0207] A linearized pGL3-TATA-EREx5-BSD was introduced, respectively,into the HeLa cells with a linearized plasmid encoding a human mutantERα or human normal ERα, as shown in Table 6 below. The linearizedplasmids were introduced into the HeLa cells by a lipofection methodusing lipofectamine (Life Technologies). According with the manualprovided with the lipofectamine, the conditions for each of thetreatments under the lipofection method included 5 hours of treatment, 7μg/dish of the plasmids (3.5 μg each) and 21 μl/dish of lipofectamine.TABLE 6 linearized plasmids first HeLa cells pGL3-TATA-EREx5-BSD andpRc/RSV-hERαKozak second HeLa cells pGL3-TATA-EREx5-BSD andpRc/RSV-hERαG390D Kozak third HeLa cells pGL3-TATA-EREx5-BSD andpRc/RSV-hERαS578P Kozak fourth HeLa cells pGL3-TATA-EREx5-BSD andpRc/RSV-hERαG390D/S578P Kozak

[0208] After the lipofection treatment, the DMEM mediums were exchangedwith DMEM medium containing 10% FBS and the transformed HeLa cells werecultured for about 36 hours. Next, the transformed HeLa cells wereremoved and collected, respectively, from the dishes by trypsintreatment and were transferred into a container containing a medium towhich blasticidin S and G418 was added thereto. The concentration of theblasticidin S therein for each of the cell cultures was 16 μg/ml. Theconcentration of the G418 therein for each of the cell cultures was 800μg/ml. The transformed HeLa cells were cultured in such mediumcontaining blasticidin S and G418 for 1 month while exchanging themedium every 3 or 4 days to a fresh batch of the medium containingblasticidin S and G418.

[0209] The resulting clones, which were able to proliferate to adiameter of from 1 to several mm, were transferred, respectively, to thewells of 96-well ViewPlates (Berthold) to which medium had previouslybeen dispensed thereto. The clones were further cultured. When theclones proliferated to such a degree that they covered 50% or more ofthe bottom surface of the well (about 5 days after the transfer), theclones were removed and collected by trypsin treatment. Each of theclones then were divided into 3 subcultures. For each of the clones, oneof the subcultures was transferred to a 96-well ViewPlate, which wasdesignated as the master plate. The other two subcultures weretransferred, respectively, to 96-well ViewPlates, which were designatedas the assay plates. The master plate and the assay plates containedmedium so that the clones can be cultured. The master plate iscontinuously cultured under similar conditions. To each of thesubcultures in the first assay plate, a DMSO solution containing 17β-E2was added to a concentration of 10 nM. An equivalent volume of DMSO wasadded to the subcultures in the second assay plate. The first and secondassay plates were then cultured for 2 days.

[0210] The medium was then removed from the wells of the first andsecond assay plates and the clones attached to the well walls werewashed twice with PBS(−). A 5-fold diluted lysis buffer PGC50 (Toyo Ink)was added to the clones in the wells of the first and second assayplates at 20 μl per well. The first and second assay plates were leftstanding at room temperature for 30 minutes and were set on aluminometer LB96P (Berthold), which was equipped with an automaticsubstrate injector. Subsequently, 50 μl of the substrate solution PGL100(Toyo Ink) was automatically dispensed, respectively, to each of thelysed clones in the assay plates to measure the luciferase activitytherein with the luminometer LB96P. Clones in master plate correspondingto the clones in the first assay plate which exhibited a 2-fold higherinduction of luciferase activity (%) were then selected as the stablytransformed ERE binary cell which stably contain the reporter gene withthe human mutant ERαG390D, S578P or G390D/S578P gene or the human normalERα gene.

6.5. Example 5 Reporter Assays of Human Mutant ERα

[0211] 6.5.1 Preparation of the Stably Transformed ERE Binary Cells forthe Reporter Assay

[0212] About 2×10⁴ cells of the stably transformed ERE binary cells,produced in the above 6.4., were then transferred to the wells of96-well Luminometer plates (Corning Coaster) to culture overnight thestably transformed ERE binary cells in an E-MEM medium to which acharcoal dextran treated FBS was added to a concentration of 10% (v/v)(hereinafter referred to as charcoal dextran FBS/E-MEM medium).

[0213] 6.5.2. Introduction of the Plasmids Encoding the Human MutantERαK303R, S309F, M396V, G415V, G494V or K531E

[0214] Seven subcultures which contained, respectively,, approximately2×10⁶ cells of the stably transformed ERE cassette cells produced in6.3., were cultured for 1 day using dishes having a diameter of about 10cm (Falcon) in charcoal dextran FBS/E-MEM medium.

[0215] For transient expression, the plasmid pRc/RSV-hERαKozak (producedin 6.1.1., encoding normal ERα) and a plasmid encoding the mutant ERα(produced in 6.1.2.2., i.e., pRc/RSV-hERαK303R Kozak, pRc/RSV-hERαS309FKozak, pRc/RSV-hERαM396V Kozak, pRc/RSV-hERαG415V Kozak,pRc/RSV-hERαG494V Kozak or pRc/RSV-hERαK531E Kozak, each encoding ahuman mutant ERα) were introduced, respectively, into the subcultures ofthe stably transformed ERE cassette cells by a lipofection method usinglipofectamine (Life Technologies). According with the manual providedwith the lipofectamine, the conditions for each of the treatments underthe lipofection method included 5 hours of treatment, 7 μg/dish of theplasmids and 21 μl/dish of lipofectamine. After culturing the resultingcell subcultures at 37° C. for 16 hours in the presence of 5% CO₂, thecharcoal dextran FBS/E-MEM medium therein was exchanged to fresh batchesof the charcoal dextran FBS/E-MEM medium to further culture each of thecell subcultures for 3 hours. The cell subcultures were then collected,respectively, and uniformly suspended in charcoal dextran FBS/E-MEMmedium.

[0216] 6.5.3. Measurement of the Activity for Transactivation of theReporter Gene

[0217] Four (4) general types of DMSO solutions were used to expose thecells in the subcultures prepared in the above 6.5.1. and 6.5.2. withvarious concentrations of a pure or partial anti-estrogen. The firstDMSO solutions were prepared to contain a partial anti-estrogen(4-hydroxytamoxifen or raloxifene) at various concentrations. The secondDMSO solutions were prepared to contain a pure anti-estrogen (ZM1 89154)at various concentrations. The third DMSO solutions were prepared tocontain E2 at 10 nM and a partial anti-estrogen (4-hydroxytamoxifen orraloxifene) at various concentrations. The fourth DMSO solutions wereprepared to contain E2 at 10 nM and a pure anti-estrogen (ZM189154) atvarious concentrations.

[0218] The first, second, third or fourth DMSO solution was then addedto the subcultures prepared, in the above 6.5.1. and 6.5.2., as shown inTables 7, 8, 9 and 10 below. The first, second, third or fourth DMSOsolution was added to the wells of the 96-well ViewPlates such that theconcentration of the first, second, third or fourth DMSO solution ineach of the wells was about 0.1% (v/v). Further, 2 controls wereprepared for each of the subcultures in the wells of a 96-wellViewPlate. One of the controls was exposed to DMSO (containing nopartial or pure anti-estrogen). The second control was exposed to a DMSOsolution consisting essentially of 100 pM of E2.

[0219] The cells were then cultured for 36 to 40 hours at 37° C. in thepresence of 5% CO₂. A 5-fold diluted lysis buffer PGC50 (Toyo Ink) wasadded, respectively, to the cells in the wells at 50 μl per well. The96-well ViewPlates were periodically and gently shook while beingincubated at room temperature for 30 minutes. Ten microliters (10 μl) ofthe lysed cells were then transferred, respectively, to white 96-wellsample plates (Berthold) and were set on a luminometer LB96P (Berthold),which was equipped with an automatic substrate injector. Subsequently,50 μl of the substrate solution PGL100 (Toyo Ink) was automaticallydispensed, respectively, to each of the lysed cells in the white 96-wellsample plates to instantaneously measure for 5 seconds the luciferaseactivity therein with the luminometer LB96P.

[0220] The luciferase activities resulting from the cells prepared in6.5.2. are illustrated in FIGS. 1 to 32.

[0221]FIGS. 1 and 2 illustrate the luciferase activity provided by thehuman normal ERα and the human mutant ERαK303R in the presence of4-hydroxytamoxifen or ZM189154 as the sole possible agent of stimulatingthe human normal ERα or the human mutant ERαK303R.

[0222]FIG. 3 illustrates the luciferase activity provided by the humannormal ERα and the human mutant ERαK303R in the presence of E2 withZM189154.

[0223]FIGS. 4 and 5 illustrate the luciferase activity provided by thehuman normal ERα and the human mutant ERαS309F in the presence of4-hydroxytamoxifen or ZM189154 as the sole possible agent of stimulatingthe human normal ERα or the human mutant ERαS309F.

[0224]FIG. 6 illustrates the luciferase activity provided by the humannormal ERα and the human mutant ERαS309F in the presence of E2 withZM189154.

[0225]FIGS. 7 and 8 illustrate the luciferase activities provided by thehuman normal ERα and the human mutant ERαM396V in the presence of4-hydroxytamoxifen or raloxifene as the sole possible agent ofstimulating the human normal ERα or the human mutant ERαM396V.

[0226] FIGS. 9 to 11 illustrate the luciferase activity provided by thehuman normal ERα and the human mutant ERαM396V in the presence of E2with 4-hydroxytamoxifen, raloxifene or ZM189154.

[0227]FIGS. 12 and 13 illustrate the luciferase activity provided by thehuman normal ERα and the human mutant ERαG415V in the presence of4-hydroxytamoxifen or ZM189154 as the sole possible agent of stimulatingthe human normal ERα or the human mutant ERαG415V.

[0228]FIGS. 14 and 15 illustrate the luciferase activity provided by thehuman normal ERα and the human mutant ERαG415V in the presence of E2with 4-hydroxytamoxifen or ZM189154.

[0229] FIGS. 16 to 17 illustrate the luciferase activity provided by thehuman normal ERα and the human mutant ERαG494V in the presence of4-hydroxytamoxifen or raloxifene as the sole possible agent ofstimulating the human normal ERα or the human mutant ERαG494V.

[0230] FIGS. 18 to 20 illustrate the luciferase activity provided by thehuman normal ERα and the human mutant ERαG494V in the presence of E2with 4-hydroxytamoxifen, raloxifene or ZM189154.

[0231] FIGS. 21 to 26 illustrate the luciferase activity provided by thehuman normal ERα and the human mutant ERαK531E in the presence of4-hydroxytamoxifen, raloxifene or ZM189154 as the sole possible agent ofstimulating the human normal ERα or the human mutant ERαK531E.

[0232] FIGS. 27 to 32 illustrate the luciferase activity provided by thehuman normal ERα and the human mutant ERαK531E in the presence of E2with 4-hydroxytamoxifen, raloxifene or ZM189154.

[0233] The luciferase activities resulting from the cells prepared in6.5.1. are illustrated in FIGS. 33 to 48.

[0234] FIGS. 33 to 40 illustrate the luciferase activity provided by thehuman normal ERα, human mutant ERαG390D, human mutant ERαS578P and humanmutant ERαG390D/S578P in the presence of 4-hydroxytamoxifen, raloxifeneor ZM189154 as the sole probable agent of stimulating the human normalERα, human mutant ERαG390D, human mutant ERαS578P and human mutantERαG390D/S578P.

[0235] FIGS. 41 to 48 illustrate the luciferase activity provided by thehuman normal ERα, human mutant ERαG390D, human mutant ERαS578P and humanmutant ERαG390D/S578P in the presence of E2 with 4-hydroxytamoxifen,raloxifene or ZM189154. TABLE 7 utilized plasmid for human normal DMSOexposed partial or or mutant ERα solution pure anti-estrogen 1pRC/RSV-hERαKozak first 4-hydroxytamoxifen 2 pRC/RSV-hERaαK303R Kozakfirst 4-hydroxytamoxifen 3 pRG/RSV-hERαKozak second ZM189154 4pRG/RSV-hERαK303R Kozak second ZM189154 5 pRC/RSV-hERαKozak fourthZM189154 6 pRC/RSV-hERαK303R Kozak fourth ZM189154 7 pRC/RSV-hERαKozakfirst 4-hydroxytamoxifen 8 pRG/RSV-hERαS309F Kozak first4-hydroxytamoxifen 9 pRC/RSV-hERαKozak second ZM189154 10pRC/RSV-hERαS309F Kozak second ZM189154 11 pRC/RSV-hERαKozak fourthZM189154 12 pRC/RSV-hERαS309F Kozak fourth ZM189154

[0236] TABLE 8 utilized plasmid for human normal DMSO exposed partial oror mutant ERα solution pure anti-estrogen 13 pRC/RSV-hERαKozak first4-hydroxytamoxifen 14 pRC/RSV-hERαM396V Kozak first 4-hydroxytamoxifen15 pRC/RSV-hERαKozak first raloxifene 16 pRC/RSV-hERαM396V Kozak firstraloxifene 17 pRC/RSV-hERαKozak third 4-hydroxytamoxifen 18pRC/RSV-hERαM396V Kozak third 4-hydroxytamoxifen 19 pRC/RSV-hERαKozakthird raloxifene 20 pRC/RSV-hERαM396V Kozak third raloxifene 21pRC/RSV-hERαKozak fourth ZM189154 22 pRC/RSV-hERαM396V Kozak fourthZM189154 23 pRC/RSV-hERαKozak first 4-hydroxytamoxifen 24pRC/RSV-hERαG415V Kozak first 4-hydroxytamoxifen 25 pRC/RSV-hERαKozaksecond ZM189154 26 pRC/RSV-hERαG415V Kozak second ZM189154 27pRC/RSV-hERαKozak third 4-hydroxytamoxifen 28 pRC/RSV-hERαG415V Kozakthird 4-hydroxytamoxifen 29 pRC/RSV-hERαKozak fourth ZM189154 30pRC/RSV-hERαG415V Kozak fourth ZM189154

[0237] TABLE 9 utilized plasmid for human normal DMSO exposed partial oror mutant ERα solution pure anti-estrogen 31 pRC/RSV-hERαKozak first4-hydroxytamoxifen 32 pRC/RSV-hERαG494V Kozak first 4-hydroxytamoxifen33 pRC/RSV-hERαKozak first raloxifene 34 pRC/RSV-hERαG494V Kozak firstraloxifene 35 pRC/RSV-hERαKozak third 4-hydroxytamoxifen 36pRC/RSV-hERαG494V Kozak third 4-hydroxytamoxifen 37 pRC/RSV-hERαKozakthird raloxifene 38 pRC/RSV-hERαG494V Kozak third raloxifene 39pRC/RSV-hERαKozak fourth ZM189154 40 pRC/RSV-hERαG494V Kozak fourthZM189154 41 pRC/RSV-hERαKozak first 4-hydroxytamoxifen 42pRC/RSV-hERαK531E Kozak first 4-hydroxytamoxifen 43 pRC/RSV-hERαKozakfirst raloxifene 44 pRC/RSV-hERαK531E Kozak first raloxifene 45pRC/RSV-hERαKozak second ZM189154 46 pRC/RSV-hERαK531E Kozak secondZM189154 47 pRC/RSV-hERαKozak third 4-hydroxytamoxifen 48pRC/RSV-hERαK531E Kozak third 4-hydroxytamoxifen 49 pRC/RSV-hERαKozakthird raloxifene 50 pRC/RSV-hERαK531E Kozak third raloxifene 51pRC/RSV-hERαKozak fourth ZM189154 52 pRC/RSV-hERαK531E Kozak fourthZM189154

[0238] TABLE 10 human normal or mutant ERα DMSO exposed partial orencoded in the chromosomes solution pure anti-estrogen 53 human normalERα first 4-hydroxytamoxifen 54 human mutant ERαG390D first4-hydroxytamoxifen 55 human mutant ERαS578P first 4-hydroxytamoxifen 56human mutant ERαG390D/S578P first 4-hydroxytamoxifen 57 human normal ERαfirst raloxifene 58 human mutant ERαG390D/S578P first raloxifene 59human normal ERα second ZM189154 60 human mutant ERαG390D/S578P secondZM189154 61 human normal ERα third 4-hydroxytamoxifen 62 human mutantERα390D third 4-hydroxytamoxifen 63 human mutant ERαS578P third4-hydroxytamoxifen 64 human mutant ERαG390D/S578P third4-hydroxytamoxifen 65 human normal ERα third raloxifene 66 human mutantERαG390D/S578P third raloxifene 67 human normal ERα fourth ZM189154 68human mutant ERαG390D/S578P fourth ZM189154

6.6. Example 6 Comparative Dually Transient Reporter Assay

[0239] Approximately 2×10⁶ HeLa cells were cultured for 1 day usingdishes having a diameter of about 10 cm (Falcon) in charcoal dextranFBS/E-MEM medium at 37° C. in the presence of 5% CO₂. After culturingthe HeLa cells, the HeLa cells were divided into two subcultures.

[0240] Subsequently, 3.75 μg of pRc/RSV-hERαKozak and 3.75 μg ofpGL3-TATA-EREx5 were introduced into the HeLa cells in the firstsubculture by a lipofection method using lipofectamine for transientexpression. In the second subculture, 3.75 μg of pRc/RSV-hERαK53 1EKozak and 3.75 μg of pGL3-TATA-EREx5 were introduced by the lipofectionmethod using lipofectamine for transient expression. The first andsecond subcultures were then cultured at 37° C. for 16 hours in thepresence of 5% CO₂. After exchanging the charcoal dextran FBS/E-MEMmedium therein with a fresh batch of charcoal dextran FBS/E-MEM mediumthe first and second subcultures were then similarly cultured for 3hours. The cells in the first and second subcultures were thencollected, respectively, and were uniformly suspended in charcoaldextran FBS/E-MEM medium.

[0241] Two (2) general types of DMSO solutions were prepared to exposethe cells in the first and second subcultures. The first DMSO solutionswere prepared to contain 4-hydroxytamoxifen at various concentrations.The second DMSO solutions were prepared to contain 10 nM of E2 and4-hydroxytamoxifen at various concentrations.

[0242] The first and second DMSO solutions were then nixed,respectively, with the first and second subcultures in 96-wellViewPlates such that the concentration of the first or second DMSOsolution in each of the wells was about 0.1% (v/v).

[0243] The first and second subcultures were then cultured for 36 hoursat 37° C. in the presence of 5% CO₂. A 5-fold diluted lysis buffer PGC50(Nippon Gene) was added, respectively, to the first and secondsubcultures in the wells at 50 μl per well. The 96-well ViewPlates wereperiodically and gently shook while being incubated at room temperaturefor 30 minutes. Ten microliters (10 μl) of the resulting lysed cellswere then transferred, respectively, to white 96-well sample plates(Berthold) and were set on a luminometer LB96P (Berthold), which wasequipped with an automatic substrate injector. Subsequently, 50 μl ofthe substrate solution PGL100 (Toyo Ink) was automatically dispensed,respectively, to each of the lysed cells in the white 96-well sampleplates to instantaneously measure for 5 seconds the luciferase activitytherein with the luminometer LB96P.

[0244] The luciferase activity from the dually transient reporter assayare shown in FIGS. 49 to 52.

[0245]FIGS. 49 and 50 illustrate the luciferase activity provided by thehuman normal ERα and human mutant ERαK531E in the presence of4-hydroxytamoxifen as the sole probable agent of stimulating the humannormal ERα or human mutant ERαK531E.

[0246]FIGS. 51 and 52 illustrate, respectively, the luciferase activityof mutant human normal ERα and human mutant ERαK531E in the presence ofE2 with 4-hydroxytamoxifen.

6.7. Example 7 Search Oligonucleotides

[0247] In order to search for a variant codon that encodes a substitutedamino acid in a human test ERα, search oligonucleotides are designed sothat the search oligonucleotides can anneal to a searching region in ahuman test ERα gene when the human test ERα gene encodes a human normalERα. The searching regions include the codon encoding the amino acid atrelative position 303, the codon encoding the amino acid at relativeposition 309, the codon encoding the amino acid at relative position390, the codon encoding the amino acid at relative position 396, thecodon encoding the amino acid at relative position 415, the codonencoding the amino acid at relative position 494, the codon encoding theamino acid at relative position 531 or the codon encoding the amino acidat relative position 578. Further the oligonucleotides are designed tohave a GC content of from 30% to 70% and a size of 20 bp. Based on theoligonuleotides so designed, the oligonucleotides of the presentinvention indicated by the above are synthesized with a DNA synthesizer(Model 394, Applied Biosystems).

6.8 Example 8 Genotype Diagnosis by PCR Amplification and NucleotideSequencing Methods

[0248] A test human liver tissue sample is used to diagnose the genotypeof the test ERα polynucleotide therein. In utilizing the test humanliver tissue sample, 0.1 g of the test human liver tissue sample ishomogenized with a homogenizer in 5 ml of a buffer containing 4Mguanidium thiocyanate, 0.1M Tris-HCl (pH 7.5) and 1% β mercaptoethanol.The resulting buffer is layered with 25 ml of an aqueous 5.7M CsClsolution and is ultracentrifuged at 90,000×g for 24 hours to obtain aRNA pellet. After rinsing the RNA pellet with 70% ethanol, the RNApellet is allowed to air dry at room temperature. The RNA pellet is thendissolved in 10 μl of sterile water to a concentration of 1.2 μg/ml. Asolution of test cDNAs is then produced by collectively using the RNAsin the RNA solution as a template in a reverse transcription reaction.In producing the test cDNAs, Superscript II (Gibco) was used with 1 μlof the RNA solution, oligo-dT oligonucleotides (Amerscham-Pharmacia) andthe buffer provided with the oligo-dT oligonucleotides. The reversetranscription reaction was allowed to react for 1 hour at 37° C.

[0249] Using 1/50 by volume samples of the test cDNAs, a PCRamplification is conducted with combinations of the searcholigonucleotides shown in Table 11 below. TABLE 11 SearchOligonucleotides 1 SEQ ID:32 and SEQ ID:38 2 SEQ ID:42 and SEQ ID:48 3SEQ ID:52 and SEQ ID:58 4 SEQ ID:62 and SEQ ID:68 5 SEQ ID:72 and SEQID:78 6 SEQ ID:82 and SEQ ID:88 7 SEQ ID:92 and SEQ ID:98 8 SEQ ID:109and SEQ ID:110

[0250] The PCR mixtures in these PCR amplifications contain the testcDNAs, AmpliTaq DNA polymerase (Perkin Elmer), b 100 μM of dNTPs (dATP,dTTP, dGTP, dCTP), one of the combinations of the searcholigonucleotides and the buffer provided with the AmpliTaq Polymerase.In this PCR amplification, there are repeated 35 times for each of thePCR amplifications, an incubation cycle entailing an incubation at 95°C. for 1 minute, then an incubation at 55° C. for 30 sec, which isfollowed by an incubation at 72° C. for 1 minute. The obtained searchingregion polynucleotides are subjected to 1% low melting point agarose gelelectrophoresis (Agarose L, Nippon Gene) and are recovered. Using wholeamounts of the recovered searching region polynucleotides, the searchingregions are sequenced. The nucleotide sequences of the searching regionsare compared to the nucleotide sequence encoding human normal ERα.

6.9. Example 9 Genotype Diagnosis by SSCP Methods

[0251] 6.9.1. Extraction of Test Genomic DNAs From a Test Tissue Sample

[0252] Test genomic DNAs from a test tissue sample is prepared by themethods described in TAKARA PCR Technical news No.2, Takara Shuzo(September 1991). This procedure in relation with the present inventionis described below.

[0253] Two (2) to 3 hair samples from a test subject are washed withsterile water and then 100% ethanol. After air drying the hair samples,the hair samples are cut to 2 to 3 mm and are transferred to a plastictest tube. Two hundred microliters (200 μl) of BCL buffer (10 mMTris-HCl (pH. 7.5), 5 mM MgCl₂, 0.32M sucrose, 1% Triton X-100) areadded thereto. Subsequently, a Proteinase K solution and a SDS solutionare mixed therewith to amount to 100 μg/ml and 0.5% (w/v), respectively.

[0254] After incubating the resulting mixture for 1 hour at 70° C., themixture is phenol-chloroform extracted to recover the aqueous layertherefrom. In the phenol-chloroform extraction, a substantially equalvolume of phenol-chloroform is added to the mixture. The mixture isshaken vigorously and is centrifuged (15,000 rpm, 20,000×g, 5 min, 4°C.). The aqueous layer therefrom is extracted with a pipette so that thephenol layer is not disturbed. A second phenol-chloroform extraction isthen similarly conducted with the aqueous layer.

[0255] A substantially equal volume of chloroform is mixed with theaqueous layer from the second phenol-chloroform extraction, to extractthe aqueous layer from the resulting chloroform mixture. In thisextraction with chloroform, the chloroform mixture is shaken vigorouslyand is centrifuged, so that the aqueous layer can be extracted from thechloroform mixture. Five hundred microliters (500 μl) of 100% ethanol isthen added to the aqueous layer from the chloroform mixture. The testgenomic DNAs therein is precipitated at −80° C. for 20 minutes and isthen centrifuged to obtain a pellet of the test genomic DNAs. Theobtained pellet of the test genomic DNAs is dried and dissolved insterile water, to so that test genomic DNAs can provide a test ERαpolynucleotide.

[0256] Alternatively, peripheral blood can be used as a test sample fromwhich test genomic DNAs can be obtained. Ten milliliters (10 ml) ofblood is collected from a test subject and test genomic DNAs areextracted from the blood, using a DNA Extraction kit (Stratagene).

[0257] 6.9.2. Analysis of Test Genomic DNA by the PCR-SSCP Method

[0258] Combinations of a forward search oligonucleotide and a reversesearch oligonucleotide are selected for PCR amplifications with the testgenomic DNAs. The combinations of the forward and reverse searcholigonucleotide are selected, based on the locus of the searchingregions in the test ERα polynucleotide. The combinations of the forwardand reverse search oligonucleotides in connection with the searchingregions which are suspected to contain the valiant codon encoding asubstituted amino acid at the provided relative positions are shown inTable 12 below. TABLE 12 searching Forward search Reverse search regionoligonucleotide oligonucleotide relative SEQ ID:29, SEQ ID:30, SEQID:34, SEQ ID:35, position 303 SEQ ID:31, SEQ ID:32 or SEQ ID:36, SEQID:37 or SEQ ID:33 SEQ ID:38 relative SEQ ID:39, SEQ ID:40, SEQ ID:44,SEQ ID:45, position 309 SEQ ID:41, SEQ ID:42 or SEQ ID:46, SEQ ID:47 orSEQ ID:43 SEQ ID:48 relative SEQ ID:49, SEQ ID:50, SEQ ID:54, SEQ ID:55,position 390 SEQ ID:51, SEQ ID:52 or SEQ ID:56, SEQ ID:57 or SEQ ID:53SEQ ID:58 relative SEQ ID:59, SEQ ID:60, SEQ ID:64, SEQ ID:65, position396 SEQ ID:61, SEQ ID:62 or SEQ ID:66, SEQ ID:67 or SEQ ID:63 SEQ ID:68relative SEQ ID:69, SEQ ID:70, SEQ ID:74, SEQ ID:75, position 415 SEQID:71, SEQ ID:72 or SEQ ID:76, SEQ ID:77 or SEQ ID:73 SEQ ID:78 relativeSEQ ID:79, SEQ ID:80, SEQ ID:84, SEQ ID:85, position 494 SEQ ID:81, SEQID:82 or SEQ ID:86, SEQ ID:87 or SEQ ID:83 SEQ ID:88 relative SEQ ID:89,SEQ ID:90, SEQ ID:94, SEQ ID:95, position 531 SEQ ID:91, SEQ ID:92 orSEQ ID:96, SEQ ID:97 or SEQ ID:93 SEQ ID:98 relative SEQ ID:99, SEQID:100, SEQ ID:104, SEQ position 578 SEQ ID:101, SEQ ID:102 ID:105, SEQID:106, or SEQ ID:103 SEQ ID:107 or SEQ ID:108

[0259] The combinations of the forward and reverse searcholigonucleotides are synthesized with a DNA synthesizer. Each of theforward and reverse search oligonucleotides are labeled with ³²P using aDNA MEGALABEL kit (Takara Shuzo). The test genomic DNAs are then used,respectively, in the PCR amplifications to provide amplified searchingregion polynucleotides. Each of the PCR mixtures in these PCRamplifications contain Amplitaq DNA Polymerase (Perkin Elmer), 400 μM ofdNTPs (100 μM of dATP, 100 μM of dTTP, 100 μM of dGTP and 100 μM ofdCTP), 100 pmol of the ³²P labeled forward search oligonucleotide, 100pmol of the ³²P labeled reverse search oligonucleotide, 1 μg of the testgenonic DNA and the buffer provided with the Amplitaq DNA Polymerase. Ineach of these PCR amplifications, there are repeated 35 times for eachof the PCR amplifications, an incubation cycle entailing an incubationat 94° C. for 1 minute, then an incubation at 55° C. for 30 seconds,which is followed by an incubation at 72° C. for 1 minute.

[0260] After the PCR amplifications, 1/20 by volume samples from each ofthe amplified searching region polynucleotides are heat denatured in 80%fomamide at 80° C. for 5 minutes. Subsequently, each the heat denaturedsearching region polynucleotides are subjected to electrophoresis in 5%native polyacrylamide gels using 180 mM Tris-borate buffer (pH 8.0). Theconditions for electrophoresis include a room temperature air coolingand a constant power of 40W for 60 min. After electrophoresis, the 5%native polyacrylamide gels are autoradiograplhed using X-ray films byusing conventional procedures to detect the radioactivity of thesearching regions.

[0261] Since a product encoding the valiant codon has a differentmobility in the 5% native polyacrylamide gel as compared with a productencoding a normal codon, a comparison of each of the mobilities of thesearching region polynucleotides with a standard polynucleotide encodinga corresponding region in a human normal ERα detects the presence orabsence of a mutation in the searching regions.

[0262] 6.9.3. Determination of Mutation

[0263] After detecting a variant codon in the searching regions, 1 mmsquare portions containing the searching region polynucleotides are cutout of the 5% native polyacrylamide gels. Each of the 1 mm squareportions are treated at 90° C. for 10 min in 100 μl of sterile water torecover the searching region polynucleotides from the 1 mm squareportions. Subsequently, 1/20 by volume samples of the searching regionpolynucleotides are then used, respectively, in a second round of PCRamplifications. The oligonucleotides in these PCR amplifications usedthe combinations of the search oligonucleotides used in the above 6.9.2.Each of the PCR mixtures in these PCR amplifications contain AmplitaqDNA Polymerase (ABI), 400 μM of dNTPs (100 μM of dATP, 100 μM of dTTP,100 μM of dGTP and 100 μM of dCTP), the forward search oligonucleotide,the reverse search oligonucleotide, one of the test DNA fragments andthe buffer provided with the Amplitaq DNA polymerase. In each of thesePCR amplifications, there are repeated 35 times, an incubation cycleentailing an incubation at 94° C. for 1 minute, then an incubation at55° C. for 30 seconds, which is followed by an incubation at 72° C. for1 minute.

[0264] After completion of the reaction, the amplified searching regionpolynucleotides are subjected to low melting point agarose gelelectrophoresis (Agarose L: Nippon Gene). After recovering the amplifiedsearching region polynucleotides from the low melting point agarosegels, the recovered searching region polynucleotides are prepared with aDye Terminator cycle sequence ready reaction kit (Applied Biosystems).The prepared sample of the searching region polynucleotides aresequenced, respectively, with an ABI autosequencer (Model 377, AppliedBiosystems), to determine the mutation in the valiant codons, ifpresent, in the searching regions.

6.10. Example 10 Genotype Diagnosis by RFLP Methods

[0265] Combinations of a forward search oligonucleotide and a reversesearch oligonucleotide are selected for PCR amplifications with testgenomic DNAs or a test cDNAs. The combinations of the forward andreverse search oligonucleotides are selected, based on the locus of thesearching regions in the test ERα polynucleotide. The combinations ofthe forward and reverse search oligonucleotides are shown in Table 13below, in connection with the searching regions which are suspected tocontain a variant codon encoding a substituted amino acid at theprovided relative positions in Table 13 below. TABLE 13 Searching regionoligonucleotides Relative position 303 SEQ ID:164 and SEQ ID:165Relative position 309 SEQ ID:166 and SEQ ID:167 Relative position 396SEQ ID:168 and SEQ ID:169 Relative position 415 SEQ ID:170 and SEQID:171 Relative position 494 SEQ ID:172 and SEQ ID:173 Relative position531 SEQ ID:174 and SEQ ID:175

[0266] The test genomic DNAs or test cDNAs are used in the PCRamplifications to provide amplified searching region polynucleotideshaving a size of about 100 or 160 bp. Each of the PCR mixtures in thesePCR amplifications contain Amplitaq DNA Polymerase (ABI), the testgenomic DNAs or test cDNAs, dNTPs (dATP, dTTP, dGTP and dCTP), theforward search oligonucleotide, the reverse search oligonucleotide andthe buffer provided with the Amplitaq DNA Polymerase. In each of thesePCR amplifications, there are repeated 35 times, an incubation cycleentailing an incubation at 94° C. for 1 minute, then an incubation at55° C. for 30 seconds, which is followed by an incubation at 72° C. for1 minute.

[0267] Samples of each of the searching region polynucleotides are thenmixed, respectively, with various restriction enzymes for restrictiondigestion reactions (one restriction enzyme per restriction digestionreaction) and are incubated at 37° C. at 1 hour. The restrictiondigestion reaction mixtures are subjected to agarose gel electrophoresisto confirm whether the searching region polynucleotides are successfullyrestriction digested with one of the various restriction enzymes. Asuccessful restriction digest with the restriction enzymes shown inTable 14 and Table 15 below indicate whether there is in the searchingregion, a valiant codon encoding a substituted amino acid at theprovided relative position in Table 14 and Table 15 below TABLE 14relative relative position 309 position 494 restriction enzyme Apa I StuI approximate length of searching region 100 bp 150 bp when encodingnormal codon restriction digestion yes yes approximate length ofresulting DNA fragments 40 bp/60 bp 100 bp/50 bp

[0268] In reference to Table 14, an unsuccessful restriction digestionwith the provided restriction enzyme at the codon encoding the aminoacid at the provided relative position, indicates that such a codon is avaliant codon. In such cases, the searching regions are sequenced withan ABI autosequencer (Model 377, Applied Biosystems) to determine themutation in the variant codons, if present, in the searching regions.TABLE 15 relative relative relative relative position position positionposition 303 396 415 531 restriction enzyme: Stu I ApaL I Kpn I Sac Iapproximate length of 100 bp 100 bp 100 bp 100 bp searching region: whenencoding normal codon restriction digestion no no no no approximatelength of — — — — resulting DNA fragments: when encoding variant codonwhen variant codon AGG GTG GTA GAG sequence is: restriction digestion:yes yes yes yes approximate length of 40 bo/ 40 bo/ 40 bo/ 40 bo/resulting DNA fragments: 60 bp 60 bp 60 bp 60 bp

[0269] In reference to Table 15, a successful restriction digestion withthe provided restriction enzyme at the codon encoding the amino acid atthe provided relative position, indicates that such a codon is a valiantcodon. In such cases, it is determined that the mutations in the variantcodons, if present, are the nucleotide sequences provided in the aboveTable 15.

6.11. Example 11 Genotype Diagnosis by Southern Hybridization Methods

[0270] Five micrograms (5 μg) of test genomic DNA, provided in 6.9.1.,is thoroughly restriction digested with the restriction enzyme Stu I.The restriction digestion reaction mixture is subjected toelectrophoresis at 20V for 16 hours with a 4% Nusieve 3:1 agarose gel(FMC BIO). The capillary alkali blotting method (Hybond blottingmembrane manual, Amerscham) is used to blot for 2 hours a nylon membranewith the separated DNA fragments in the 4% Nuseive 3:1 agarose gel tothe nylon membrane. Followed by lightly washing the blotted filter with2×SSC buffer (0.3M NaCl, 0.33M Na-Citrate, pH 7.0), the blotted nylonmembrane is dried at 80° C. for 90 minutes.

[0271] The blotted nylon membrane is treated at 55° C. for 16 hours withprehybridization buffer (6×SSPE (0.9M NaCl, 0.052M NaH₂PO₄, 7.5 mMEDTA), 0.5% SDS, 5×Denhart and 0.1 mg/ml of salmon sperm DNA). Theprehybridization buffer is then exchanged with an equal volume ofhybridization buffer (6×SSPE (0.9M NaCl, 0.052M NaH₂PO₄, 7.5 mM EDTA),0.5% SDS, 5×Denhart, 0.1 mg/ml of salmon sperm DNA and a ³²P labeledprobe oligonucleotide). In the hybridization buffer, the radioactiveconcentration of the ³²P labeled probe oligonucleotide is at least10×10⁸ cpm for every 150 ml of the hybridization buffer. As the ³²Plabeled probe oligonucleotide, there is utilized the oligonucleotidedepicted in SEQ ID: 81 which is labeled with ³²P at the ends thereof.The ³²P labeled probe is produced by incubating at 37° C. for 1 hourwith γ ³²P-ATP, T4 polynucleotide kinase and 1 μg of the oligonucleotidedepicted in SEQ ID: 81 in the buffer provided with the T4 polynucleotidekinase.

[0272] After the hybridization, the blotted nylon membrane is washedtwice with washing buffer containing 1×SSC (0.15 M NaCl, 15 mM sodiumcitrate) and 0.5% SDS. In washing the blotting filter twice, the blottednylon membrane is incubated after each washing at 62° C. for 40 minutesin the washing buffer.

[0273] The blotted membrane is then autoradiographed for 10 days withx-ray film to analyze whether the restriction enzyme Stu I is successfulin restriction digesting at the restriction site therein overlappingwith the codon in the searching region which is suspected to be avariant codon encoding a substituted amino acid at relative position494. A successful restriction digest with the restriction enzyme Stu Iindicates that there is in the test ERα polynucleotide, a nucleotidesequence encompassing AGGCCT, overlapping with the codon encoding theamino acid at relative position 494. In such cases, it is determinedthat the test ERα is a normal ERα. An unsuccessful restriction digestwith the restriction enzyme Stu I at the corresponding locus, indicatesthat there is in the test ERα polynucleotide, a variant codon encoding asubstituted amino acid at relative position 494. In such cases, thesearching region is sequenced with an ABI autosequencer (Model 377,Applied Biosystems), to determine the mutation in the variant codon, ifpresent, in the searching region.

6.12. Example 12 Production of a Plasmid Encoding Human Normal AR

[0274] A human prostate cDNA library (CLONETECH, Quick clonecDNA#7123-1) is utilized to PCR amplify therefrom a cDNA encoding ahuman normal AR (Genbank Accession No. M23263). The PCR mixture in thisPCR amplification contains 10 ng of the human prostate cDNA library, 10pmol of an oligonucleotide depicted in SEQ ID: 176, 10 pmol of anoligonucleotide depicted in SEQ ID: 177, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). The oligonucleotides depicted in SEQ ID: 176 and SEQID: 177 are synthesized with a DNA synthesizer (Model 394, AppliedBiosystems, ). In this PCR amplification, there is repeated 35 timeswith a PCRsystem 9700 (Applied Biosystems), an incubation cycleentailing an incubation at 95° C. for 1 minute followed by an incubationat 68° C. for 3 minutes.

[0275] The resulting PCR mixture is subjected to low melting pointagarose gel electrophoresis (Agarose L: Nippon Gene) to confirm withethidium bromide staining, that the cDNA encoding the human normal AR isPCR amplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

[0276] Another PCR amplification is then conducted to add a Kozakconsensus sequence immediately upstream from the stair codon (ATG) inthe cDNA. The PCR mixture in this PCR amplification contains 100 ng ofthe cDNA encoding the human normal AR, an oligonucleotide depicted inSEQ ID: 178 and an oligonucleotide depicted in SEQ ID: 179, LA-TaqPolymerase (Takara Shuzo), the buffer provided with the LA-TaqPolymerase and dNTPs (dATP, dTTP, dGTP, dCTP). In this PCRamplification, there is repeated 25 times an incubation cycle entailingan incubation at 95° C. for 1 minute followed by an incubation at 68° C.for 3 minutes. The resulting PCR mixture is subjected to low meltingpoint agarose gel electrophoresis (Agarose L: Nippon Gene). Afterrecovering the amplified cDNA from the low melting point agarose gel, 1μg of the amplified cDNA is treated with a DNA Blunting Kit (TakaraShuzo) to blunt the ends of the amplified cDNA. Subsequently, theresulting cDNA therefrom is allowed to react with a T4 polynucleotidekinase to phosphorylate the ends thereof. After phenol treating thephosphorylated cDNA, the phosphorylated cDNA is ethanol precipitated toachieve a purified form of the phosphoylated cDNA.

[0277] The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV isthen treated with a DNA Blunting Kit (Takara Shuzo) to blunt the endsthereof and is subjected to low melting point agarose gelelectrophoresis (Agarose L, Nippon Gene). After recovering therestriction digested pRc/RSV from the low melting point agarose gel, 100ng of the restriction digested pRc/RSV and all of the above purifiedform of the phosphorylated cDNA are used in a ligation reaction with aT4 DNA ligase. The ligation reaction mixture is used to transform E.coli competent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB-amp. The clones thereof showing an ampicillin resistanceare then recovered. Some of the clones are then used to isolatetherefrom the plasmids derived from the ligation reaction. An aliquotsample of each of the isolated plasmids is then prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The isolated plasmidsare sequenced with an ABI autosequencer (Model 377, Applied Biosystems),to confirm that there is a plasmid encoding the human normal AR. Such aplasmid is selected and is designated as pRc/RSV-hAR Kozak.

6.13. Example 13 Production of a Plasmid Encoding a Human Normal GR

[0278] A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1)is utilized to PCR amplify therefrom a cDNA encoding a normal GR(Genbank Accession No. M10901). The PCR mixture in this PCRamplification contains 10 ng of the human liver cDNA library, 10 pmol ofan oligonucleotide depicted in SEQ ID: 180, 10 pmol of anoligonucleotide depicted in SEQ ID: 181, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). The oligonucleotides depicted in SEQ ID: 180 and SEQID: 181 are synthesized with a DNA synthesizer (Model 394, AppliedBiosystems). In this PCR amplification, there is repeated 35 times witha PCRsystem 9700 (Applied Biosystems), an incubation cycle entailing anincubation at 95° C. for 1 minute followed by an incubation at 60° C.for 3 minutes.

[0279] The resulting PCR mixture is subjected to low melting pointagarose gel electrophoresis (Agarose L: Nippon Gene) to confirm withethidium bromide staining, that the cDNA encoding the human normal GR isPCR amplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

[0280] Another PCR amplification is then conducted to add a Kozakconsensus sequence immediately upstream from the start codon (ATG) inthe cDNA. The PCR mixture in this PCR amplification contains 100 ng ofthe cDNA encoding the human normal GR, an oligonucleotide depicted inSEQ ID: 182 and an oligonucleotide depicted in SEQ ID: 183, LA-TaqPolymerase (Takara Shuzo), the buffer provided with the LA-TaqPolymerase and dNTPs (dATP, dTTP, dGTP, dCTP). In this PCRamplification, there is repeated 25 times an incubation cycle entailingan incubation at 95° C. for 1 minute followed by an incubation at 60° C.for 3 minutes. The resulting PCR mixture is subjected to low meltingpoint agarose gel electrophoresis (Agarose L: Nippon Gene). Afterrecovering the amplified cDNA from the low melting point agarose gel, 1μg of the amplified cDNA is treated with a DNA Blunting Kit (TakaraShuzo) to blunt the ends of the amplified cDNA. Subsequently, theresulting cDNA therefrom is allowed to react with a T4 polynucleotidekinase to phosphorylate the ends of the cDNA. After phenol treating thephosphorylated cDNA, the phosphorylated cDNA is ethanol precipitated toachieve a purified form of th,e phosphoylated cDNA.

[0281] The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated cDNA are used in a ligation reaction with a T4 DNAligase. The reaction mixture is used to transform E. coli competent DH5αcells (TOYOBO). The transformed E. coli cells are cultured in LB-amp.The clones thereof showing an ampicillin resistance are then recovered.Some of the clones are then used to isolate therefrom the plasmidsderived from the ligation reaction. An aliquot sample of each of theisolated plasmids are then prepared with a Dye Terminator Sequence KitFS (Applied Biosystems). The isolated plasmids are sequenced with an ABIautosequencer (Model 377, Applied Biosystems), to confirm that there isa plasmid encoding the normal GR. The plasmid is selected and isdesignated as pRc/RSV-hGR Kozak.

6.14. Example 14 Production of a Plasmid Encoding a Human Normal PR

[0282] A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1)is utilized to PCR amplify therefrom a cDNA encoding a normal PR(Genbank Accession No. M15716). The PCR mixture in this PCRamplification contains 10 ng of the human liver cDNA library, 10 pmol ofa oligonucleotide depicted in SEQ ID: 184, 10 pmol of a oligonucleotidedepicted in SEQ ID: 185, LA-Taq Polymerase (Takara Shuzo), the bufferprovided with the LA-Taq Polymerase and dNTPs (dATP, dTTP, dGTP, dCTP).The oligonucleotides depicted in SEQ ID: 184 and SEQ ID: 185 aresynthesized with a DNA synthesizer (Model 394, Applied Biosystems). Inthis PCR amplification, there is repeated 35 times with a PCRsystem 9700(Applied Biosystems), an incubation cycle entailing an incubation at 95°C. for 1 minute followed by an incubation at 55° C. for 3 minutes.

[0283] The resulting PCR mixture is subjected to low melting pointagarose gel electrophoresis (Agarose L: Nippon Gene) to confirm withethidium bromide staining, that the cDNA encoding human normal PR is PCRamplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

[0284] Another PCR amplification is then conducted to add a Kozakconsensus sequence immediately upstream from the start codon (ATG) inthe cDNA. The PCR mixture in this PCR amplification contains 100 ng ofthe cDNA encoding normal PR, a oligonucleotide depicted in SEQ ID: 186and a oligonucleotide depicted in SEQ ID: 187, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). In this PCR amplification, there is repeated 25 timesan incubation cycle entailing an incubation at 95° C. for 1 minutefollowed by an incubation at 55° C. for 3 minutes. The resulting PCRmixture is subjected to low melting point agarose gel electrophoresis(Agarose L: Nippon Gene). After recovering the amplified test cDNA fromthe low melting point agarose gel, 1 μg of the amplified test cDNA istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends of theamplified test cDNA. Subsequently, the resulting test cDNA therefrom isallowed to react with a T4 polynucleotide kinase to phosphorylate theends of the cDNA. After phenol treating the phosphorylated test cDNA,the phosphorylated test cDNA is ethanol precipitated to achieve apurified form of the phosphoylated test cDNA.

[0285] The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated test cDNA are used in a ligation reaction with a T4 DNAligase. The ligation reaction mixture is used to transform E. colicompetent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB-amp. The clones thereof showing an ampicillin resistanceare then recovered. Some of the clones are then used to isolatetherefrom the plasmids derived from the ligation reaction. An aliquotsample of each of the isolated plasmids are then prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The isolated plasmidsare sequenced with an ABI autosequencer (Model 377, Applied Biosystems),to confirm that there is a plasmid encoding normal PR. Such a plasmid isselected and is designated as pRc/RSV-hPR Kozak.

6.15. Example 15 Production of a Plasmid Encoding a Human Normal MR

[0286] A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1)is utilized to PCR amplify therefrom a cDNA encoding a normal MR(Genbank Accession No. M16801). The PCR mixture in this PCRamplification contains 10 ng of the human liver cDNA library, 10 pmol ofan oligonucleotide depicted in SEQ ID: 188, 10 pmol of anoligonucleotide depicted in SEQ ID: 189, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). The oligonucleotides depicted in SEQ ID: 188 and SEQID: 189 are synthesized with a DNA synthesizer (Model 394, AppliedBiosystems). In this PCR amplification, there is repeated 35 times witha PCRsystem 9700 (Applied Biosystems), an incubation cycle entailing anincubation at 95° C. for 1 minute followed by an incubation at 60° C.for 3 minutes.

[0287] The resulting PCR mixture is subjected to low melting pointagarose gel electrophoresis (Agarose L: Nippon Gene) to confirm withethidium bromide staining, that the cDNA encoding the normal MR is PCRamplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

[0288] Another PCR amplification is then conducted to add a Kozakconsensus sequence immediately upstream from the start codon (ATG) inthe cDNA. The PCR mixture in this PCR amplification contains 100 ng ofthe cDNA encoding the normal MR, a oligonucleotide depicted in SEQ ID:190 and a oligonucleotide depicted in SEQ ID: 191, LA-Taq Polymerase(Takara Shuzo), the buffer provided with the LA-Taq Polymerase and dNTPs(dATP, dTTP, dGTP, dCTP). In this PCR amplification, there is repeated25 times an incubation cycle entailing an incubation at 95° C. for 1minute followed by an incubation at 60° C. for 3 minutes. The resultingPCR mixture is subjected to low melting point agarose gelelectrophoresis (Agarose L: Nippon Gene). After recovering the amplifiedcDNA from the low melting point agarose gel, 1 μg of the amplified cDNAis treated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends ofthe amplified cDNA. Subsequently, the resulting cDNA therefrom isallowed to react with a T4 polynucleotide kinase to phosphorylate theends of the cDNA. After phenol treating the phosphorylated cDNA, thephosphorylated cDNA is ethanol precipitated to achieve a purified formof the phosphoylated cDNA.

[0289] The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated cDNA are used in a ligation reaction with a T4 DNAligase. The ligation reaction mixture is used to transform E. colicompetent DH5α cells (TOYOBO). The transformed E.coli cells are culturedin LB-amp. The clones thereof showing an ampicillin resistance are thenrecovered. Some of the clones are then used to isolate therefrom theplasmids derived from the ligation reaction. An aliquot sample of eachof the isolated plasmids are then prepared with a Dye TerminatorSequence Kit FS (Applied Biosystems). The isolated plasmids aresequenced with an ABI autosequencer (Model 377, Applied Biosystems), toconfirm that there is a plasmid encoding normal MR. The plasmid isselected and is designated as pRc/RSV-hMR Kozak.

6.16. Example 16 Production of a Stably Transformed Cell Which StablyContains in One of Its Chromosomes the MMTV Reporter Gene

[0290] The plasmid pMSG (Pharmacia) is restriction digested withrestriction enzymes Hind III and Sma I to provide a DNA fragmentencoding a partial sequence of the MMTV-LTR region, which has a size of1463 bp. The 1463 bp DNA fragment is then treated with a DNA BluntingKit (Takara Shuzo) to blunt the ends of the 1463 bp DNA fragment.

[0291] The plasmid pGL3 (Promega), which encodes the firefly luciferasegene, is restriction digested with restriction enzymes Bgl II and HindIII and is then treated with BAP at 65° C. for 1 hour. The restrictiondigestion reaction mixture is then subjected to low melting pointagarose gel electrophoresis (Agarose L, Nippon Gene) to confirm thatthere is a DNA fragment having a nucleotide sequence encoding thefirefly luciferase. The DNA fragment having the nucleotide sequenceencoding the firefly luciferase is then recovered from the low meltingpoint agarose gel. Subsequently, 100 ng of the recovered DNA fragmenthave the nucleotide sequence encoding firefly luciferase and 1 μg of the1463 bp DNA fragment are used in a ligation reaction with T4 DNA ligase.The ligation reaction mixture is then used to transform E. colicompetent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB-amp. The clones thereof showing an ampicillin resistanceare then recovered. Some of the clones are then used to isolatetherefrom the plasmids derived from the ligation reaction. An aliquotsample of each of the isolated plasmids are then restriction digestedwith restriction enzymes Kpn I and Cla I. The restriction digestionreaction mixtures are subjected to agarose gel electrophoresis toconfirm that there is a plasmid which contains 1 copy of the 1463 bp DNAfragment operably upstream from the DNA fragment have the nucleotidesequence encoding firefly luciferase (hereinafter referred to as theMMTV reporter gene). Such a plasmid is selected and is designated aspGL3-MMTV.

[0292] The plasmid pUCSV-BSD (Funakoshi) is restriction digested withrestriction enzyme BamH I to prepare a DNA encoding a blasticidin Sdeaminase gene expression cassette. Further, the plasmid pGL3-MMTV isrestriction digested with restriction enzyme BamH I and is then treatedwith BAP at 65° C. for 1 hour. The resulting DNA encoding theblasticidin S deaminase gene expression cassette and the restrictiondigested pGL3-MMTV are mixed together to be used in a ligation reactionwith T4 DNA ligase. The ligation reaction mixture is used to transformE. coli competent DH5α cells. The transformed E. coli cells are culturedin LB-amp. The clones thereof showing an ampicillin resistance are thenrecovered. Some of the clones are then used to isolate therefrom theplasmids derived from the ligation reaction. An aliquot sample of eachof the isolated plasmids are then prepared with a Dye TerminatorSequence Kit FS (Applied Biosystems). The isolated plasmids aresequenced with an ABI autosequencer (Model 377, Applied Biosystems) toconfirm there is a plasmid which has a structure in which the DNAencoding a blasticidin S Deaminase gene expression cassette has beeninserted into the Bam HI restriction site in pGL3-MMTV. Such a plasmidis selected and is designated as pGL3-MMTV-BSD.

[0293] In order to produce stably transformed cells which stably containin one of its chromosomes the MMTV reporter gene (hereinafter referredto as the stably transformed MMTV cassette cell), the plasmidpGL3-MMTV-BSD is linearized and introduced into HeLa cells.

[0294] The plasmid pGL3-MMTV-BSD is restriction digested withrestriction enzyme Sal I to linearize pGL3-MMTV-BSD.

[0295] Approximately 5×10⁵ HeLa cells were cultured as host cells for 1day using dishes having a diameter of about 10 cm (Falcon) in DMEMmedium (Nissui Pharmaceutical Co.) containing 10% FBS at 37° C. underthe presence of 5% CO₂.

[0296] The linearized pGL3-MMTV-BSD is then introduced to the culturedHeLa cells by a lipofection method using lipofectamine (LifeTechnologies). According with the manual provided with thelipofectamine, the conditions under the lipofection method included 5hours of treatment, 7 μg/dish of the linearized pGL3-MMTV-BSD and 21μl/dish of lipofectamine.

[0297] After the lipofection treatment, the DMEM medium is exchangedwith DMEM medium containing 10% FBS and the transformed HeLa cells arecultured for about 36 hours. Next, the transformed HeLa cells areremoved and collected from the dish by trypsin treatment and aretransferred into a container containing a medium to which blasticidin Sis added to a concentration of 16 μg/ml. The transformed HeLa cells arecultured in such medium containing blasticidin S for 1 month whileexchanging the medium every 3 or 4 days to a fresh batch of the mediumcontaining blasticidin S.

[0298] The resulting clones, which are able to proliferate and produce acolony having a diameter of from 1 to several mm, are transferred as awhole to the wells of a 96-well ViewPlate (Berthold) to which medium ispreviously dispensed thereto. The colonies of the clones are furthercultured. When the clones proliferated to such a degree that theycovered 50% or more of the bottom surface of each of the wells (about 5days after the transfer), the clones are removed and collected bytrypsin treatment. The clones then are divided into 2 subcultures. Oneof the subcultures is transferred to a 96-well ViewPlate, which isdesignated as the master plate. The other subculture is transferred to a96-well ViewPlate, which is designated as the assay plate. The masterplate and the assay plate contain medium so that the clones can becultured. The master pate is continuously cultured under similarconditions.

[0299] The medium is then removed from the wells of the assay plate andthe clones attached to the well walls are washed twice with PBS(−). A5-fold diluted lysis buffer PGC50 (Toyo Ink) is added, respectively, tothe clones in the wells of the assay plate at 20 μl per well. The assayplate is left standing at room temperature for 30 minutes and is set ona luminometer LB96P (Berthold), which is equipped with an automaticsubstrate injector. Subsequently, 50 μl of the substrate solution PGL100(Toyo Ink) is automatically dispensed to the lysed clones in the assayplate to measure the luciferase activity therein with the luminometerLB96P. A plurality of the clones, which exhibited a high luciferaseactivity are selected therefrom.

[0300] Samples of the selected clones are then cultured at 37° C. for 1to 2 weeks in the presence of 5% CO₂ using dishes having a diameter ofabout 10 cm (Falcon) in charcoal dextran FBS/E-MEM medium.

[0301] The plasmid pRc/RSV-hAR Kozak is then introduced to the samplesof the selected clones by a lipofection method using lipofectamine (LifeTechnologies) to provide a second round of clones. According with themanual provided with the lipofectamine, the conditions under thelipofection method included 5 hours of treatment, 7 μg/dish of theplasmids above and 21 μl/dish of lipofectamine. A DMSO solutioncontaining dihydrotestosterone (DHT), which is the natural cognateligand of a normal AR, is then added to the resulting second clones sothat the concentration of DHT in the medium is 10 nM. After culturingthe second clones for 2 days, the luciferase activity is measured,similarly to the above, for each of the second clones. The clone in themaster plate, which provided the second clone exhibiting the highestinduction of luciferase activity, is selected as the stably transformedMMTV cassette cell.

[0302] In this regard, the stably transformed MMTV cassette cell can beused in reporter assays with AR, GR, PR, MR and the like.

6.17. Example 17 Reporter Assay of the Human Normal AR as a Human TestAR

[0303] 6.17.1. Preparation of Stably Transformed MMTV Cassette Cell

[0304] Approximately 2×10⁶ stably transformed MMTV cassette cellsprovided in 6.16., are cultured at 37° C. for 1 day in the presence of5% CO₂ using dishes having a diameter of about 10 cm (Falcon) incharcoal dextran FBS/E-MEM medium.

[0305] For transient expression, the plasmid pRc/RSV-hAR Kozak isintroduced into a subculture of the stably transformed MMTV cassettecells by a lipofection method using lipofectamine (Life Technologies).According with the manual provided with the lipofectamine, theconditions under the lipofection method include 5 hours of treatment, 7μg/dish of the pRc/RSV-hAR Kozak and 21 μl/dish of lipofectamine. Afterculturing the resulting cell subculture at 37° C. for 16 hours in thepresence of 5% CO₂, the charcoal dextran FBS/E-MEM medium therein isexchanged to fresh batches of the charcoal dextran FBS/E-MEM medium tofurther culture the cell subculture for 3 hours. The cell subculture isthen collected and uniformly suspended in charcoal dextran FBS/E-MEMmedium to provide a subculture thereof.

[0306] 6.17.2. Measurement of the Activity for Transactivation of theMMTV Reporter Gene

[0307] First DMSO solutions are prepared to contain variousconcentrations of flutamide. The flutamide is used in the first DMSOsolution as an agonist directed to the normal AR. Further, second DMSOsolutions are prepared to contain 10 nM of DHT and the variousconcentrations of the flutamide. The flutamide is used in the secondDMSO solution as an antagonist directed to the normal AR.

[0308] The first and second DMSO solutions are then mixed, respectively,with the subcultures prepared in the above 6.17.1., in the 96-wellViewPlates such that the concentration of the first or second DMSOsolution in each of the wells is about 0.1% (v/v). Further, as astandard, a sample of the cell subculture which is provided in 6.17.1.,is nixed with a DMSO solution containing DHT, in the wells of a96-ViewPlate.

[0309] The cells are then cultured for 40 hours at 37° C. in thepresence of 5% CO₂. A 5-fold diluted lysis buffer PGC50 (Toyo Ink) isadded, respectively, to the subcultures in the wells at 50 μl per well.The 96-well ViewPlates are periodically and gently shook while beingincubated at room temperature for 30 minutes. Ten microliters (10 μl) ofthe lysed cells are then transferred, respectively, to white 96-wellsample plates (Berthold) and are set on a luminometer LB96P (Berthold),which is equipped with an automatic substrate injector. Subsequently, 50μl of the substrate solution PGL100 (Toyo Ink) is automaticallydispensed, respectively, to each of the lysed cells in the white 96-wellsample plates to instantaneously measure for 5 seconds the luciferaseactivity therein with the luminometer LB96P.

[0310] Further, the above reporter assay can use as the test AR, amutant AR. In this regard, a plasmid encoding a mutant AR is usedinstead of pRc/RSV-hAR Kozak. To provide the plasmid encoding the mutantAR, a Kozak consensus sequence is added operably upstream from apolynucleotide encoding a mutant AR and the resulting polynucleotide isinserted into a restriction site of Hind III in the plasmid pRc/RSV(Invitrogen), as similarly described above.

6.18. Example 18 Reporter Assay of a Human Normal GR as the Human TestGR

[0311] 6.18.1. Preparation of Stably Transformed MMTV Cassette Cell

[0312] Approximately 2×10⁶ stably transformed MMTV cassette cellsprovided in 6.16., are cultured at 37° C. for 1 day in the presence of5% CO₂ using dishes having a diameter of about 10 cm (Falcon) incharcoal dextran FBS/E-MEM medium.

[0313] For transient expression, the plasmid pRc/RSV-hGR Kozak isintroduced into a subculture of the stably transformed MMTV cassettecells by a lipofection method using lipofectamine (Life Technologies).According with the manual provided with the lipofectamine, theconditions under the lipofection method include 5 hours of treatment, 7μg/dish of the pRc/RSV-hAR Kozak and 21 μl/dish of lipofectamine. Afterculturing the resulting cell subculture at 37° C. for 16 hours in thepresence of 5% CO₂, the charcoal dextran FBS/E-MEM medium therein isexchanged to fresh batches of the charcoal dextran FBS/E-MEM medium tofurther culture the cell subculture for 3 hours. The cell subculture isthen collected and uniformly suspended in charcoal dextran FBS/E-MEMmedium.

[0314] 6.18.2. Measurement of the Activity for Transactivation of theMMTV Reporter Gene

[0315] First DMSO solutions are prepared to contain variousconcentrations of pregnanolone 16α carbonitrile (PCN). The PCN is usedin the first DMSO solutions as an agonist with the normal GR. Further,the second DMSO solutions are prepared to contain 10 nM ofcorticosterone and the various concentrations of PCN. The PCN is used inthe second DMSO solutions as an antagonist with the normal GR.

[0316] The first and second DMSO solutions are then mixed, respectively,with the cell subcultures prepared in the above 6.18.1., in wells of the96-well ViewPlates such that the concentration of the first or secondDMSO solution in each of the wells is about 0.1% (v/v). Further, as astandard, a sample of the cell subculture is mixed with a DMSO solutioncontaining corticosterone in the wells of a 96-well ViewPlate.

[0317] The cells are then cultured for 40 hours at 37° C. in thepresence of 5% CO₂. A 5-fold diluted lysis buffer PGC50 (Toyo Ink) isadded, respectively, to the subcultures in the wells at 50 μl per well.The 96-well ViewPlates are periodically and gently shook while beingincubated at room temperature for 30 minutes. Ten microliters (10 μl) ofthe lysed cells are then transferred, respectively, to white 96-wellsample plates (Berthold) and are set on a luminometer LB96P (Berthold),which is equipped with an automatic substrate injector. Subsequently, 50μl of the substrate solution PGL100 (Toyo Ink) is automaticallydispensed, respectively, to each of the lysed cells in the white 96-wellsample plates to instantaneously measure for 5 seconds the luciferaseactivity therein with the luminometer LB96P.

[0318] Further, the above reporter assay can use as the test GR, amutant GR. In this regard, a plasmid encoding the mutant GR is usedinstead of pRc/RSV-hGR Kozak. To provide the plasmid encoding the mutantGR, a Kozak consensus sequence is added operably upstream from apolynucleotide encoding a mutant GR and the resulting polynucleotide isinserted into a restriction site of Hind III in the plasmid pRc/RSV(Invitrogen), as similarly described above.

6.19. Example 19 Reporter Assay of Human Normal PR as the Human Test PR

[0319] 6.19.1. Preparation of Stably Transformed MMTV Cassette Cell

[0320] Approximately 2×10⁶ stably transformed MMTV cells provided in6.16., are cultured at 37° C. for 1 day in the presence of 5% CO₂ usingdishes having a diameter of about 10 cm (Falcon) in charcoal dextranFBS/E-MEM medium.

[0321] For transient expression the plasmid pRc/RSV-hPR Kozak isintroduced into a subculture of the stably transformed MMTV cassettecells by a lipofection method using lipofectamine (Life Technologies).According with the manual provided with the lipofectamine, theconditions under the lipofection method include 5 hours of treatment, 7μg/dish of the pRc/RSV-hAR Kozak and 21 μl/dish of lipofectamine. Afterculturing the resulting cell subculture at 37° C. for 16 hours in thepresence of 5% CO₂, the charcoal dextran FBS/E-MEM medium therein isexchanged to fresh batches of the charcoal dextran FBS/E-MEM medium tofurther culture each of the cell subculture for 3 hours. The cellsubculture is then collected and uniformly suspended in charcoal dextranFBS/E-MEM medium.

[0322] 6.19.2. Measurement of the Activity for Transactivation of theMMTV Reporter Gene

[0323] First DMSO solutions are prepared to contain variousconcentrations of RU486. The RU486 is used in the first DMSO solutionsas an agonist directed to the normal PR. Further, second DMSO solutionsare prepared to contain 10 nM of progesterone and the variousconcentrations of RU486. The RU486 is used in the second DMSO solutionsas an antagonist directed to the normal PR.

[0324] The first and second DMSO solutions are mixed, respectively, withthe cell subcultures prepared in the above 6.19.1., in the 96-wellViewPlates such that the concentration of the first or second DMSOsolution in each of the wells is about 0.1% (v/v). Further, as astandard, a sample of the cell subculture is mixed with a DMSO solutioncontaining progesterone.

[0325] The cells are then cultured for 40 hours at 37° C. in thepresence of 5% CO₂. A 5-fold diluted lysis buffer PGC50 (Toyo Ink) isadded, respectively, to the cells in the wells at 50 μl per well. The96-well ViewPlates are periodically and gently shook while beingincubated at room temperature for 30 minutes. Ten microliters (10 μl) ofthe lysed cells are then transferred, respectively, to white 96-wellsample plates (Berthold) and are set on a luminometer LB96P (Berthold),which is equipped with an automatic substrate injector. Subsequently, 50μl/well of the substrate solution PGL100 (Toyo Ink) is automaticallydispensed, respectively, to each of the lysed cells in the white 96-wellsample plates to instantaneously measure for 5 seconds the luciferaseactivity therein with the luminometer LB96P.

[0326] Further, the above reporter assay can use as the test PR, amutant PR. In this regard, a plasmid encoding the mutant PR is usedinstead of pRc/RSV-hPR Kozak. To provide the plasmid encoding the mutantPR, a Kozak consensus sequence is added operably upstream from apolynucleotide encoding a mutant PR and the resulting polynucleotide isinserted into a restriction site of Hind III in the plasmid pRc/RSV(Invitrogen), as similarly described above.

6.20. Example 20 Production of a Plasmid Encoding Human Normal ERβ

[0327] A human prostate cDNA library (CLONETECH, Quick clonecDNA#7123-1) is utilized to PCR amplify therefrom a cDNA encoding ahuman normal ERβ (Genbank Accession No. AB006590). The PCR mixture inthis PCR amplification contains 10 ng of the human liver cDNA library,10 pmol of an oligonucleotide depicted in SEQ ID: 192, 10 pmol of anoligonucleotide depicted in SEQ ID: 193, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). The oligonucleotides depicted in SEQ ID: 192 and SEQID: 193 are synthesized with a DNA synthesizer (Model 394, AppliedBiosystems). In this PCR amplification, there is repeated 35 times witha PCRsystem 9700 (Applied Biosystems), an incubation cycle entailing anincubation at 95° C. for 1 minute followed by an incubation at 68° C.for 3 minutes.

[0328] The resulting PCR mixture is subjected to low melting pointagarose gel electrophoresis (Agarose L: Nippon Gene) to confirm withethidium bromide staining, that the cDNA encoding human normal ERβ isPCR amplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

[0329] Another PCR amplification is then conducted to add a Kozakconsensus sequence immediately upstream from the start codon (ATG) inthe cDNA. The PCR mixture in this PCR amplification contains 100 ng ofthe cDNA, 10 pmol of an oligonucleotide depicted in SEQ ID: 194 and 10pmol of an oligonucleotide depicted in SEQ ID: 195, LA-Taq Polymerase(Takara Shuzo), the buffer provided with the LA-Taq Polymerase and dNTPs(dATP, dTTP, dGTP, dCTP). In this PCR amplification, there is repeated25 times an incubation cycle entailing an incubation at 95° C. for 1minute followed by an incubation at 68° C. for 3 minutes. The resultingPCR mixture is subjected to low melting point agarose gelelectrophoresis (Agarose L: Nippon Gene). After recovering the amplifiedcDNA from the low melting point agarose gel, 1 μg of the amplified cDNAis treated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends ofthe amplified cDNA. Subsequently, the resulting cDNA therefrom isallowed to react with a T4 polynucleotide kinase to phosphorylate theends of the cDNA. After phenol treating the phosphorylated cDNA, thephosphorylated cDNA is ethanol precipitated to achieve a purified formof the phosphoylated cDNA.

[0330] The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated cDNA are used in a ligation reaction with a T4 DNAligase. The ligation reaction mixture is used to transform E. colicompetent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB-amp. The clones thereof showing an ampicillin resistanceare then recovered. Some of the clones are then used to isolatetherefrom the plasmids derived from the ligation reaction. An aliquotsample of the isolated plasmids are then prepared with a Dye TerminatorSequence Kit FS (Applied Biosystems). The isolated plasmids aresequenced with an ABI autosequencer (Model 377, Applied Biosystems), toconfirm that there is a plasmid encoding human normal ERβ. Such aplasmid is selected and is designated as pRc/RSV-hERβ Kozak.

6.21. Example 21 Reporter Assay of Human Normal ERβ as the Human TestERβ

[0331] 6.21.1. Preparation of Stably Transformed ERE Cassette Cell

[0332] Approximately 2×10⁶ stably transformed ERE cassette cellsprovided in 6.3., are cultured at 37° C. for 1 day in the presence of 5%CO₂ using dishes having a diameter of about 10 cm (Falcon) in charcoaldextran FBS/E-MEM medium.

[0333] For transient expression, the plasmid pRc/RSV-hERβ Kozak isintroduced into a subculture of the stably transformed ERE cassettecells by a lipofection method using lipofectamine (Life Technologies).According with the manual provided with the lipofectamine, theconditions under the lipofection method include 5 hours of treatment, 7μg/dish of the pRc/RSV-hERβ Kozak and 21 μl/dish of lipofectamine. Afterculturing the resulting cell subculture at 37° C. for 16 hours in thepresence of 5% CO₂, the charcoal dextran FBS/E-MEM medium therein isexchanged to fresh batches of the charcoal dextran FBS/E-MEM medium tofurther culture the cell subculture for 3 hours. The cell subculture isthen collected and uniformly suspended in charcoal dextran FBS/E-MEMmedium.

[0334] 6.21.2. Measurement of the Activity for Transactivation of theERE Reporter Gene

[0335] First DMSO solutions are prepared to contain variousconcentrations of 4-hydroxytamoxifen. The 4-hydroxytamoxifen is used inthe first DMSO solutions as an agonist directed to the human normal ERα.Further, second DMSO solutions are prepared to contain 10 nM of E2 andthe various concentrations of 4-hydroxytamoxifen. The 4-hydroxytamoxifenis used in the second DMSO solutions as an antagonist directed to theERβ.

[0336] The first and second DMSO solutions are mixed, respectively, withthe subcultures prepared in the above 6.21.1., in the 96-well ViewPlatessuch that the concentration of the first or second DMSO solution in eachof the wells is about 0.1% (v/v). Further, as a standard, a sample ofthe cells is mixed with DMSO solutions containing E2 in the wells of a96-well ViewPlate.

[0337] The cells are then cultured for 40 hours at 37° C. in thepresence of 5% CO₂. A 5-fold diluted lysis buffer PGC50 (Toyo Ink) isadded, respectively, to the cells in the wells at 50 μl per well. The96-well ViewPlates are periodically and gently shook while beingincubated at room temperature for 30 minutes. Ten microliters (10 μl) ofthe lysed cells are then transferred, respectively, to white 96-wellsample plates (Berthold) and are set on a luminometer LB96P (Berthold),which is equipped with an automatic substrate injector. Subsequently, 50μl/well of the substrate solution PGL100 (Toyo Ink) is automaticallydispensed, respectively, to each of the lysed cells in the white 96-wellsample plates to instantaneously measure for 5 seconds the luciferaseactivity therein with the luminometer LB96P.

[0338] Further, the above reporter assay can use as the test ERβ, amutant ERβ. In this regard, a plasmid encoding the mutant ERβ is usedinstead of pRc/RSV-hERβ Kozak. To provide the plasmid encoding themutant ERβ, a Kozak consensus sequence is added operably upstream from apolynucleotide encoding a mutant ERβ and the resulting polynucleotide isinserted into a restriction site of Hind III in the plasmid pRc/RSV(Invitrogen), as similarly described above.

6.22. Example 22 Production of a Plasmid Encoding a Human Normal TRα

[0339] A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1)is utilized to PCR amplify therefrom a cDNA encoding a human normal TRα(Genbank Accession No. M24748). The PCR mixture in this PCRamplification contains 10 ng of the human liver cDNA library, 10 pmol ofan oligonucleotide depicted in SEQ ID: 196, 10 pmol of anoligonucleotide depicted in SEQ ID: 197, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). The oligonucleotides depicted in SEQ ID: 196 and SEQID: 197 are synthesized with a DNA synthesizer (Model 394, AppliedBiosystems). In this PCR amplification, there is repeated 35 times witha PCRsystem 9700 (Applied Biosystems), an incubation cycle entailing anincubation at 95° C. for 1 minute followed by an incubation at 68° C.for 3 minutes.

[0340] The resulting PCR mixture is subjected to low melting pointagarose gel electrophoresis (Agarose L: Nippon Gene) to confirm withethidium bromide staining, that the cDNA encoding human normal TRα isPCR amplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

[0341] Another PCR amplification is then conducted to add a Kozakconsensus sequence immediately upstream from the start codon (ATG) inthe cDNA. The PCR mixture in this PCR amplification contains 100 ng ofthe cDNA encoding human normal TRα, 10 pmol of an oligonucleotidedepicted in SEQ ID: 198 and 10 pmol of an oligonucleotide depicted inSEQ ID: 199, LA-Taq Polymerase (Takara Shuzo), the buffer provided withthe LA-Taq Polymerase and dNTPs (dATP, dTTP, dGTP, dCTP). In this PCRamplification, there is repeated 25 times an incubation cycle entailingan incubation at 95° C. for 1 minute followed by an incubation at 68° C.for 3 minutes. The resulting PCR mixture is subjected to low meltingpoint agarose gel electrophoresis (Agarose L: Nippon Gene). Afterrecovering the amplified cDNA from the low melting point agarose gel, 1μg of the amplified cDNA is treated with a DNA Blunting Kit (TakaraShuzo) to blunt the ends of the amplified cDNA. Subsequently, theresulting cDNA therefrom is allowed to react with a T4 polynucleotidekinase to phosphorylate the ends of the cDNA. After phenol treating thephosphorylated cDNA, the phosphorylated cDNA is ethanol precipitated toachieve a purified form of the phosphorylated cDNA.

[0342] The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated cDNA are used in a ligation reaction with a T4 DNAligase. The ligation reaction mixture is used to transform E. colicompetent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB-amp. The clones thereof showing an ampicillin resistanceare then recovered. Some of the clones are then used to isolatetherefrom the plasmids derived from the ligation reaction. An aliquotsample of the isolated plasmids are then prepared with a Dye TerminatorSequence Kit FS (Applied Biosystems). The isolated plasmids aresequenced with an ABI autosequencer (Model 377, Applied Biosystems), toconfirm that there is a plasmid encoding human normal TRα. Such aplasmid is selected and is designated as pRc/RSV-hTRαKozak.

6.23. Example 23 Production of a Plasmid Encoding a Human Normal TRβ

[0343] A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1)is utilized to PCR amplify therefrom a cDNA encoding a human normal TRβ(Genbank Accession No. M26747). The PCR mixture in this PCRamplification contains 10 ng of the human liver cDNA library, 10 pmol ofan oligonucleotide depicted in SEQ ID: 200, 10 pmol of anoligonucleotide depicted in SEQ ID: 201, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). The oligonucleotides depicted in SEQ ID: 200 and SEQID: 201 are synthesized with a DNA synthesizer (Model 394, AppliedBiosystems). In this PCR amplification, there is repeated 35 times witha PCRsystem 9700 (Applied Biosystems), an incubation cycle entailing anincubation at 95° C. for 1 minute followed by an incubation at 68° C.for 3 minutes.

[0344] The resulting PCR mixture is subjected to low melting pointagarose gel electrophoresis (Agarose L: Nippon Gene) to confirm withethidium bromide staining, that the cDNA encoding human normal TRβ isPCR amplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

[0345] Another PCR amplification is then conducted to add a Kozakconsensus sequence immediately upstream from the stair codon (ATG) inthe cDNA. The PCR mixture in this PCR amplification contains 100 ng ofthe cDNA encoding normal TRα, 10 pmol of an oligonucleotide depicted inSEQ ID: 202 and 10 pmol of an oligonucleotide depicted in SEQ ID: 203,LA-Taq Polymerase (Takara Shuzo), the buffer provided with the LA-TaqPolymerase and dNTPs (dATP, dTTP, dGTP, dCTP). In this PCRamplification, there is repeated 25 times an incubation cycle entailingan incubation at 95° C. for 1 minute followed by an incubation at 68° C.for 3 minutes. The resulting PCR mixture is subjected to low meltingpoint agarose gel electrophoresis (Agarose L, Nippon Gene). Afterrecovering the amplified cDNA from the low melting point agarose gel, 1μg of the amplified cDNA is treated with a DNA Blunting Kit (TakaraShuzo) to blunt the ends of the amplified cDNA. Subsequently, theresulting cDNA therefrom is allowed to react with a T4 polynucleotidekinase to phosphorylate the ends of the cDNA. After phenol treating thephosphorylated cDNA, the phosphorylated cDNA is ethanol precipitated toachieve a purified form of the phosphoylated cDNA.

[0346] The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated cDNA are used in a ligation reaction with a T4 DNAligase. The ligation reaction mixture is used to transform E. colicompetent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB-amp. The clones thereof showing an ampicillin resistanceare then recovered. Some of the clones are then used to isolatetherefrom the plasmids derived from the ligation reaction. An aliquotsample of each of the isolated plasmids are then prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The isolated plasmidsare sequenced with an ABI autosequencer (Model 377, Applied Biosystems),to confirm that there is a plasmid encoding human normal TRβ. Such aplasmid is selected and is designated as pRc/RSV-hTRβ Kozak.

6.24. Example 24 Production of a Plasmid Containing an DR4 Reporter Gene

[0347] An oligonucleotide depicted in SEQ ID: 204 and an oligonucleotidehaving a nucleotide sequence complementary thereto are synthesized witha DNA synthesizer. The oligonucleotide depicted in SEQ ID: 204 issynthesized to encode one of the strands of an DR4. The secondoligonucleotide is synthesized to have a nucleotide sequencecomplementary to the first oligonucleotide. The two oligonucleotides areannealed together to produce a DNA encoding a DR4 sequence (hereinafterreferred to as the DR4 DNA). A T4 polynucleotide kinase is allowed toreact with the DR4 DNA to phosphorylate the ends thereof. The DR4 DNA isthen ligated together with a T4 DNA ligase to provide a DR4×5 DNA havinga 5 tandem repeat of the DR4 sequence. The ligation reaction mixture isthen subjected to low melting point agarose gel electrophoresis (AgaroseL, Nippon Gene), and the DR4×5 DNA is recovered from the gel.

[0348] The plasmid pGL3-TATA provided in 6.2., is restriction digestedwith restriction enzyme Sma I and is then treated with BAP at 65° C. for1 hour. The restriction digested reaction mixture is then subjected tolow melting point agarose gel electrophoresis (Agarose L, Nippon Gene).After recovering the DNA fragment having a nucleotide sequence encodingfirefly luciferase from the low melting point agarose gel, 100 ng of therecovered DNA fragment and 1 μg of the DR4×5 DNA are used in a ligationreaction. The resulting ligation reaction mixture is then used totransform E. coli competent DH5α cells (TOYOBO). The transformed E. colicells are cultured in LB-amp. The clones thereof showing an ampicillinresistance are then recovered. Some of the clones are then used toisolate therefrom the plasmids derived from the ligation reaction. Analiquot sample of each of the isolated plasmids are then restrictiondigested with restriction enzymes Kpn I and Xho I. The restrictiondigestion reaction mixtures are subjected to agarose gel electrophoresisto confirm that there is a plasmid having a structure in which the DR4×5DNA is inserted into the restriction site of restriction enzyme Sma I inthe pGL3-TATA. Such a plasmid is selected and is designated aspGL3-TATA-DR4×5.

[0349] The plasmid pGL3-TATA-DR4×5 is then restriction digested withrestriction enzyme Sal I. After a Blunting Kit (Takara Shuzo) is used toblunt the ends of the restriction digested pGL3-TATA-DR4×5, therestriction digested pGL3-TATA-DR4×5 is treated with BAP at 65° C. for 1hour. The Blunting Kit is also used to blunt the ends of the DNAfragment encoding the blasticidin S deaminase gene (BamH I-BamH Ifragment) provided in 6.2.

[0350] The DNA fragment encoding a blasticidin S deaminase geneexpression cassette and the restriction digested pGL3-TATA-DR4×5 arethen mixed together for a ligation reaction with T4 DNA ligase. Theligation reaction mixture is used to transform E. coli competent DH5αcells (TOYOBO). The transformed E. coli cells are cultured in LB-amp.The clones thereof showing an ampicillin resistance are then recovered.Some of the clones are then used to isolate therefrom the plasmidsderived from the ligation reaction. An aliquot sample of each of theisolated plasmids are then prepared with a Dye Terminator Sequence KitFS (Applied Biosystems). The isolated plasmids are sequenced with an ABIautosequencer (Model 377, Applied Biosystems) to confirm whether thereis a plasmid which has structure in which the DNA encoding a blasticidinS deaminase gene expression cassette is inserted into the restrictionsite of restriction enzyme Sal I in pGL3-TATA-DR4×5. Such a plasmid isselected and is designated as pGL3-TATA-DR4×5-BSD.

6.25. Example 25 Production of a Stably Transformed Cell Which StablyContains in One of Its Chromosomes the DR4 Reporter Gene

[0351] In order to produce stably transformed cells which stablycontains in one of its chromosomes the DR4 reporter gene (hereinafterreferred to as the stably transformed DR4 cassette cell, the plasmidpGL3-TATA-DR4×5-BSD was linearized and introduced into HeLa cells.

[0352] The plasmid pGL3-TATA-DR4×5-BSD is restriction digested withrestriction enzyme Not I to linearize pGL3-TATA-DR4×5-BSD.

[0353] Approximately 5×10⁵ HeLa cells are cultured as host cells for 1day using dishes having a diameter of about 10 cm (Falcon) in DMEMmedium (Nissui Pharmaceutical Co.) containing 10% FBS at 37° C. in thepresence of 5% CO₂.

[0354] The linearized pGL3-TATA-DR4×5-BSD is then introduced to thecultured HeLa cells by a lipofection method using lipofectamine (LifeTechnologies). According with the manual provided with thelipofectamine, the conditions under the lipofection method include 5hours of treatment, 7 μg/dish of the linearized pGL3-TATA-DR4×5-BSD and21 μl/dish of lipofectamine.

[0355] After the lipofection treatment, the DMEM medium is exchangedwith DMEM medium containing 10% FBS and the transformed HeLa cells arecultured for about 36 hours. Next, the transformed HeLa cells areremoved and collected from the dish by trypsin treatment and aretransferred into a container containing a medium to which blasticidin Sis added to a concentration of 16 μg/ml. The transformed HeLa cells arecultured in such medium containing blasticidin S for 1 month whileexchanging the medium containing blasticidin S every 3 or 4 days to afresh batch of the medium containing blasticidin S.

[0356] The resulting clones, which are able to proliferate and produce acolony having a diameter of from 1 to several mm, are transferred,respectively, as a whole to the wells of a 96-well ViewPlate (Berthold)to which medium is previously dispensed thereto. The clones are furthercultured. When the clones proliferated to such a degree that clonestherein covered 50% or more of the bottom surface of each of the wells(about 5 days after the transfer), the clones are removed and collectedby trypsin treatment. Each of the clones then are divided into 2subcultures. One of the subcultures is transferred to a 96-wellViewPlate, which is designated as the master plate. The other subculturewas transferred to a 96-well ViewPlate, which is designated as the assayplate. The master plate and the assay plate contain medium so that theclones can be cultured. The master pate is continuously cultured undersimilar conditions.

[0357] The medium in the wells of the assay plate is then removedtherefrom and the clones attached to the well walls are washed twicewith PBS(−). A 5-fold diluted lysis buffer PGC50 (Toyo Ink) is added,respectively, to the clones in the wells of the assay plate at 20 μl/well. The assay plate is left standing at room temperature for 30minutes and is set on a luminometer LB96P (Berthold), which is equippedwith an automatic substrate injector. Subsequently, 50 μl of thesubstrate solution PGL100 (Toyo Ink) is automatically dispensed to thelysed clones in the assay plate to measure the luciferase activitytherein with the luminometer LB96P. A plurality of the clones, whichexhibited a luciferase activity are selected therefrom.

[0358] Samples of the selected clones are then cultured at 37° C. for 1day in the presence of 5% CO₂ using dishes having a diameter of about 10cm (Falcon) in charcoal dextran FBS/E-MEM medium.

[0359] The plasmid pRc/RSV-hTRαKozak is then introduced to the samplesof the selected clones by a lipofection method using lipofectamine (LifeTechnologies) to provide a second round of clones. According with themanual provided with the lipofectamine, the conditions under thelipofection method included 5 hours of treatment, 7 μg/dish ofpRc/RSV-hTRαKozak above and 21 μl/dish of lipofectamine. A DMSO solutioncontaining triiodothyronine (T3), which is the natural cognate ligand ofa human normal TRα, is then added to the resulting second clones so thatthe concentration of T3 in the medium is 10 nM. After culturing thesecond clones for 2 days, the luciferase activity is measured, similarlyto the above, for each of the second clones. The clone in the masterplate, which provided the second clone exhibiting the highest inductionof luciferase activity, is selected as the stably transformed DR4cassette cell.

[0360] In this regard, the stably transformed DR4 cassette cell can beused in reporter assays with TRα, TRβ, CAR, LXR, PXR and the like.

6.26. Example 26 Production of a Plasmid Encoding Human Normal VDR

[0361] A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1)is utilized to PCR amplify therefrom a cDNA encoding a human normal VDR(Genbank Accession No. J03258). The PCR mixture in this PCRamplification contains 10 ng of the human liver cDNA library, 10 pmol ofan oligonucleotide depicted in SEQ ID: 205, 10 pmol of anoligonucleotide depicted in SEQ ID: 206, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). The oligonucleotides depicted in SEQ ID: 205 and SEQID: 206 are synthesized with a DNA synthesizer (Model 394, AppliedBiosystems). In this PCR amplification, there is repeated 35 times witha PCRsystem 9700 (Applied Biosystems), an incubation cycle entailing anincubation at 95° C. for 1 minute followed by an incubation at 68° C.for 3 minutes.

[0362] The resulting PCR mixture is subjected to low melting pointagarose gel electrophoresis (Agarose L, Nippon Gene) to confirm withethidium bromide staining, that the cDNA encoding human normal VDR isPCR amplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

[0363] Another PCR amplification is then conducted to add a Kozakconsensus sequence immediately upstream from the start codon (ATG) inthe cDNA encoding normal. The PCR mixture in this PCR amplificationcontains 100 ng of the cDNA encoding normal, 10 pmol of anoligonucleotide depicted in SEQ ID: 207 and 10 pmol of anoligonucleotide depicted in SEQ ID: 208, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). In this PCR amplification, there is repeated 25 timesan incubation cycle entailing an incubation at 95° C. for 1 minutefollowed by an incubation at 68° C. for 3 minutes. The resulting PCRmixture is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the amplified cDNA from thelow melting point agarose gel, 1 μg of the amplified cDNA is treatedwith a DNA Blunting Kit (Takara Shuzo) to blunt the ends of theamplified cDNA. Subsequently, the resulting cDNA therefrom is allowed toreact with a T4 polynucleotide kinase to phosphorylate the ends of thecDNA. After phenol treating the phosphorylated cDNA, the phosphorylatedcDNA is ethanol precipitated to achieve a purified form of thephosphorylated cDNA.

[0364] The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated cDNA is used in a ligation reaction with a T4 DNA ligase.The ligation reaction mixture is used to transform E. coli competentDH5α cells (TOYOBO). The transformed E. coli cells are cultured inLB-amp. The clones thereof showing an ampicillin resistance are thenrecovered. Some of the clones are then used to isolate therefrom theplasmids derived from the ligation reaction. An aliquot sample of eachof the isolated plasmids are then prepared with a Dye TerminatorSequence Kit FS (Applied Biosystems). The isolated plasmids aresequenced with an ABI autosequencer (Model 377, Applied Biosystems), toconfirm that there is a plasmid encoding human normal VDR. Such aplasmid is selected and is designated as pRc/RSV-hVDR Kozak.

6.27. Example 27 Production of a Plasmid Containing a DR3 Reporter Gene

[0365] An oligonucleotide depicted in SEQ ID: 209 and an oligonucleotidehaving a nucleotide sequence complementary thereto are synthesized witha DNA synthesizer. The oligonucleotide depicted in SEQ ID: 209 issynthesized to encode one of the strands of a DR3. The secondoligonucleotide is synthesized to have a nucleotide sequencecomplementary to the first oligonucleotide. The two oligonucleotides areannealed together to produce a DNA encoding a DR3 sequence (hereinafterreferred to as the DR3 DNA). A T4 polynucleotide kinase is allowed toreact with the DR3 DNA to phosphorylate the ends thereof. The DR3 DNA isthen ligated together with a T4 DNA ligase to provide a DR3×5 DNA havinga 5 tandem repeat of the DR3 sequence. The ligation reaction mixture isthen subjected to low melting point agarose gel electrophoresis (AgaroseL, Nippon Gene), and the DR3×5 DNA is recovered from the gel .

[0366] The plasmid pGL3-TATA provided in 6.2., is restriction digestedwith restriction enzyme Sma I and is then treated with BAP at 65° C. for1 hour. The restriction digested reaction mixture is then subjected tolow melting point agarose gel electrophoresis (Agarose L, Nippon Gene).After recovering the DNA fragment having a nucleotide sequence encodingfirefly luciferase from the low melting point agarose gel, 100 ng of therecovered DNA fragment and 1 μg of the DR3×5 DNA are used in a ligationreaction. The resulting ligation reaction mixture is then used totransform E. coli competent DH5α cells (TOYOBO). The transformed E. colicells are cultured in LB-amp. The clones thereof showing an ampicillinresistance are then recovered. Some of the clones are then used toisolate therefrom the plasmids derived from the ligation reaction. Analiquot sample of each of the isolated plasmids is then restrictiondigested with restriction enzymes Kpn I and Xho I. The restrictiondigestion reaction mixtures are subjected to agarose gel electrophoresisto confirm that there is a plasmid having a structure in which the DR3×5DNA is inserted into the restriction site of restriction enzyme Sma I inthe pGL3-TATA. Such a plasmid is selected and is designated aspGL3-TATA-DR3×5.

[0367] The plasmid pGL3-TATA-DR3x5 is then restriction digested withrestriction enzyme Sal I. After a Blunting Kit (Takara Shuzo) is used toblunt the ends of the restriction digested pGL3-TATA-DR3×5, therestriction digested pGL3-TATA-DR3×5 is treated with BAP at 65° C. for 1hour. The Blunting Kit is also used to blunt the ends of the DNAfragment having the blasticidin S deaminase gene expression cassette(BamH I-BamH I fragment) provided in 6.2. The blunt endedpGL3-TATA-DR3×5 and the blunt ended DNA fragment having the blasticidinS deaminase gene expression cassette are used in a ligation reactionwith T4 DNA ligase. The resulting ligation reaction mixture is then usedto transform E. coli competent DH5α cells (TOYOBO). The transformed E.coli cells are cultured in LB-amp. The clones thereof showing anampicillin resistance are then recovered. Some of the clones are thenused to isolate therefrom the plasmids derived from the ligationreaction. The isolated plasmid in which the DNA fragment having theblasticidin S deaminase gene expression cassette is inserted to therestriction site of restriction enzyme Sal I in pGL3-TATA-DR3×5 isselected and is designated as pGL3-TATA-DR3×5-BSD.

6.28. Example 28 Production of a Stably Transformed Cassette Cell WhichStably Contains in One of its Chromosomes the DR3 Reporter Gene

[0368] In order to produce stably transformed cells which stably containin one of its chromosomes the DR3 reporter gene (hereinafter referred toas the stably transformed DR3 cassette cell, the plasmidpGL3-TATA-DR3×5-BSD was linearized and introduced into HeLa cells.

[0369] The plasmid pGL3-TATA-DR3×5-BSD is restriction digested withrestriction enzyme Not I to linearize pGL3-TATA-DR3×5-BSD.

[0370] Approximately 5×10⁵ HeLa cells are cultured as host cells for 1day using dishes having a diameter of about 10 cm (Falcon) in DMEMmedium (Nissui Pharmaceutical Co.) containing 10% FBS at 37° C. underthe presence of 5% CO₂.

[0371] The linearized pGL3-TATA-DR3×5-BSD are then introduced to thecultured HeLa cells by a lipofection method using lipofectamine (LifeTechnologies). According with the manual provided with thelipofectamine, the conditions under the lipofection method include 5hours of treatment, 7 μg/dish of the linearized pGL3-TATA-DR3x5-BSD and21 μl/dish of lipofectamine.

[0372] After the lipofection treatment, the DMEM medium is exchangedwith DMEM medium containing 10% FBS and the transformed HeLa cells arecultured for about 36 hours. Next, the transformed HeLa cells areremoved and collected from the dish by trypsin treatment and aretransferred into a container containing a medium to which blasticidin Sis added to a concentration of 16 μg/ml. The transformed cells arecultured in such medium containing blasticidin S for 1 month whileexchanging the medium containing blasticidin S every 3 or 4 days to afresh batch of the DMEM medium containing blasticidin S.

[0373] The clones, which are able to proliferate and produce a colonyhaving a diameter of from 1 to several mm, are transferred,respectively, as a whole to the wells of a 96-well ViewPlate (Berthold)to which medium is previously dispensed thereto. The clones are furthercultured. When the clones proliferated to such a degree that eukaryoticclones therein covered 50% or more of the bottom surface of each of thewells (about 5 days after the transfer), the clones are removed andcollected by trypsin treatment. The clones then are divided into 2subcultures. One of the subcultures is transferred to a 96-wellViewPlate, which is designated as the master plate. The other subcultureis transferred to a 96-well ViewPlate, which is designated as the assayplate. The master plate and the assay plate contain medium so that theclones can be cultured. The master pate is continuously cultured undersimilar conditions.

[0374] The medium in the wells of the assay plate is then removedtherefrom and the clones attached to the well walls are washed twicewith PBS(−). A 5-fold diluted lysis buffer PGC50 (Toyo Ink) is added,respectively, to the clones in the wells of the assay plate at 20μl/well. The assay plate is left standing at room temperature for 30minutes and is set on a luminometer LB96P (Berthold), which is equippedwith an automatic substrate injector. Subsequently, 50 μl of thesubstrate solution PGL100 (Toyo Ink) is automatically dispensed to thelysed clones in the assay plate to measure the luciferase activitytherein with the luminometer LB96P. A plurality of the clones, whichexhibited a high luciferase activity are selected therefrom.

[0375] Samples of the selected clones are then cultured at 37° C. for 1to 2 weeks in the presence of 5% CO₂ using dishes having a diameter ofabout 10 cm (Falcon) in charcoal dextran FBS/E-MEM medium.

[0376] The plasmid pRc/RSV-hVDR Kozak is then introduced to the samplesof the selected clones by a lipofection method using lipofectamine (LifeTechnologies) to provide a second round of clones. According with themanual provided with the lipofectamine, the conditions under thelipofection method included 5 hours of treatment, 7 μg/dish ofpRc/RSV-VDR Kozak above and 21 μl/dish of lipofectamine. A DMSO solutioncontaining 1,25-(OH) Vitamin D₃, which is the natural cognate ligand ofa human normal VDR, is then added to the resulting second clones so thatthe concentration of 1,25-(OH) Vitamin D₃ in the medium is 10 nM. Afterculturing the second clones for 2 days, the luciferase activity ismeasured, similarly to the above, for each of the second clones. Theclone in the master plate, which provided the second clone exhibitingthe highest induction of luciferase activity, is selected as the stablytransformed DR3 cassette cell.

6.29. Example 29 Production of a Plasmid Encoding Normal PPAR γ

[0377] A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1)is utilized to PCR amplify therefrom a cDNA encoding a human normal PPARγ (Genbank Accession No. U79012). The PCR mixture in this PCRamplification contains 10 ng of the human liver cDNA library, 10 pmol ofan oligonucleotide depicted in SEQ ID: 210, 10 pmol of anoligonucleotide depicted in SEQ ID: 211, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). The oligonucleotides depicted in SEQ ID: 210 and SEQID: 211 are synthesized with a DNA synthesizer (Model 394, AppliedBiosystems). In this PCR amplification, there is repeated 35 times witha PCRsystem 9700 (Applied Biosystems), an incubation cycle entailing anincubation at 95° C. for 1 minute followed by an incubation at 68° C.for 3 minutes.

[0378] The resulting PCR mixture is subjected to low melting pointagarose gel electrophoresis (Agarose L: Nippon Gene) to confirm withethidium bromide staining, that the cDNA encoding human normal PPAR γ isPCR amplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

[0379] Another PCR amplification is then conducted to add a Kozakconsensus sequence immediately upstream from the start codon (ATG) inthe cDNA. The PCR mixture in this PCR amplification contains 100 ng ofthe cDNA encoding normal PPAR γ and Kozak consensus sequence, anoligonucleotide depicted in SEQ ID: 212 and an oligonucleotide depictedin SEQ ID: 211, LA-Taq Polymerase (Takara Shuzo), the buffer providedwith the LA-Taq Polymerase and dNTPs (dATP, dTTP, dGTP, dCTP). In thisPCR amplification, there is repeated 25 times an incubation cycleentailing an incubation at 95° C. for 1 minute followed by an incubationat 68° C. for 3 minutes. The resulting PCR mixture is subjected to lowmelting point agarose gel electrophoresis (Agarose L, Nippon Gene).After recovering the amplified cDNA from the low melting point agarosegel, 1 μg of the amplified cDNA is treated with a DNA Blunting Kit(Takara Shuzo) to blunt the ends of the amplified cDNA. Subsequently,the resulting cDNA therefrom is allowed to react with a T4polynucleotide kinase to phosphorylate the ends of the cDNA. Afterphenol treating the phosphorylated cDNA, the phosphorylated cDNA isethanol precipitated to achieve a purified form of the phosphoylatedcDNA.

[0380] The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated cDNA are used in a ligation reaction with a T4 DNAligase. The ligation reaction mixture is used to transform E. colicompetent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB-amp. The clones thereof showing an ampicillin resistanceare then recovered. Some of the clones are then used to isolatetherefrom the plasmids derived from the ligation reaction. Each ofisolated plasmids is then prepared with a Dye Terminator Sequence Kit FS(Applied Biosystems). The isolated plasmids are sequenced with an ABIautosequencer (Model 377, Applied Biosystems), to confirm that there isa plasmid encoding human normal PPAR γ. Such a plasmid is selected andis designated as pRc/RSV-hPPAR γ Kozak.

6.30. Example 30 Production of a Plasmid Containing a DR1 Reporter Gene

[0381] An oligonucleotide depicted in SEQ ID: 213 and an oligonucleotidehaving a nucleotide sequence complementary thereto are synthesized witha DNA synthesizer. The oligonucleotide depicted in SEQ ID: 213 issynthesized to encode one of the strands of a DR1 sequence. The secondoligonucleotide is synthesized to have a nucleotide sequencecomplementary to the first oligonucleotide. The two oligonucleotides areannealed together to produce a DNA encoding a DR1 sequence (hereinafterreferred to as the DR1 DNA). A T4 polynucleotide kinase is allowed toreact with the DR1 DNA to phosphorylate the ends thereof. The DR1 DNA isthen ligated together with a T4 DNA ligase to provide a DR1×5 DNA havinga 5 tandem repeat of the DR 1 sequence. The ligation reaction mixture isthen subjected to low melting point agarose gel electrophoresis (AgaroseL, Nippon Gene), and the DR1×5 DNA is recovered from the gel .

[0382] The plasmid pGL3-TATA provided in 6.2., is restriction digestedwith restriction enzyme Sma I and is then treated with BAP at 65° C. for1 hour. The restriction digestion reaction mixture is then subjected tolow melting point agarose gel electrophoresis (Agarose L, Nippon Gene).After recovering the DNA fragment having a nucleotide sequence encodingfirefly luciferase from the low melting point agarose gel, 100 ng of therecovered DNA fragment and 1 μg of the DR1×5 DNA are used in a ligationreaction with T4 DNA ligase. The resulting ligation reaction mixture isthen used to transform E. coli competent DH5α cells (TOYOBO). Thetransformed E. coli cells are cultured in LB-amp. The clones thereofshowing an ampicillin resistance are then recovered. Some of the clonesare then used to isolate therefrom the plasmids derived from theligation reaction. An aliquot sample of each of the isolated plasmids isthen restriction digested with restriction enzymes Kpn I and Xho I. Therestriction digestion reaction mixtures are subjected to agarose gelelectrophoresis to confirm that there is a plasmid in which the DR1×5DNA is inserted into the restriction site of restriction enzyme Sma I inthe pGL3-TATA. The plasmid is then sequenced with an ABI autosequencer(Model 377, Applied Biosystems), to confirm that there is provided aplasmid having a 5 tandem repeat of the DR1 sequence. Such a plasmid isselected and is designated as pGL3-TATA-DR1×5.

[0383] The plasmid pGL3-TATA-DR1×5 is then restriction digested withrestriction enzyme Sal I. After a Blunting Kit (Takara Shuzo) is used toblunt the ends of the restriction digested pGL3-TATA-DR1×5, therestriction digested pGL3-TATA-DR1×5 is treated with BAP at 65° C. for 1hour. The Blunting Kit is also used to blunt the ends of the DNAfragment encoding the blasticidin S deaminase gene (BamH I-BamH Ifragment derived from pUCSV-BSD (Funakoshi)) provided in 6.2.

[0384] The DNA fragment encoding a blasticidin S deaminase geneexpression cassette and the restriction digested pGL3-TATA-DR1×5 arethen mixed together for a ligation reaction with T4 DNA ligase. Theligation reaction mixture is used to transform E. coli competent DH5 αcells (TOYOBO). The transformed E. coli cells are cultured in LB-amp.The clones thereof showing an ampicillin resistance are then recovered.Some of the clones are then used to isolate therefrom the plasmidsderived from the ligation reaction. An aliquot sample of each of theisolated plasmids are then prepared with a Dye Terminator Sequence KitFS (Applied Biosystems). The isolated plasmids are sequenced with an ABIautosequencer (Model 377, Applied Biosystems) to confirm whether theplasmid has a structure in which the DNA encoding a blasticidin Sdeaminase gene expression cassette has been inserted into therestriction site of restriction enzyme Sal I in pGL3-TATA-DR1×5. Theplasmid is selected and is designated as pGL3-TATA-DR1×5-BSD.

6.31. Example 31 Production or a Stably Transformed Cassette Cell WhichStably Contain in One of its Chromosomes the DR1 Reporter Gene

[0385] In order to produce stably transformed cassette cells whichstably contain in one of its chromosomes the DR1 reporter gene(hereinafter referred to as the stably transformed DR1 cassette cell),the plasmid pGL3-TATA-DR1×5-BSD is linearized and introduced into HeLacells.

[0386] The plasmid pGL3-TATA-DR1×5-BSD is restriction digested withrestriction enzyme Not I to linearize pGL3-TATA-DR1×5-BSD.

[0387] Approximately 5×10⁵ HeLa cells are cultured as host cells for 1day using dishes having a diameter of about 10 cm (Falcon) in DMEMmedium (Nissui Pharmaceutical Co.) containing 10% FBS at 37° C. underthe presence of 5% CO₂.

[0388] The linearized pGL3-TATA-DR1×5-BSD is then introduced to thecultured HeLa cells by a lipofection method using lipofectamine (LifeTechnologies). According with the manual provided with thelipofectamine, the conditions under the lipofection method include 5hours of treatment, 7 μg/dish of the linearized pGL3-TATA-DR1×5-BSD and21 μl/dish of lipofectamine.

[0389] After the lipofection treatment, the DMEM medium is exchangedwith DMEM medium containing 10% FBS and the transformed HeLa cells arecultured for about 36 hours. Next, the transformed HeLa cells areremoved and collected from the dish by trypsin treatment and aretransferred into a container containing a medium to which blasticidin Sis added to a concentration of 16 μg/ml. The transformed HeLa cells arecultured in such medium containing blasticidin S for 1 month whileexchanging the medium containing blasticidin S every 3 or 4 days to afresh batch of the medium containing blasticidin S.

[0390] The clones, which are able to proliferate and produce a colonyhaving a diameter of from 1 to several mm, are transferred,respectively, as a whole to the wells of a 96-well ViewPlate (Berthold)to which medium is previously dispensed thereto. The clones are furthercultured. When the clones proliferated to such a degree that clonestherein covered 50% or more of the bottom surface of each of the wells(about 5 days after the transfer), the clones are removed and collectedby trypsin treatment. The clones then are divided into 2 subcultures.One of the subcultures is transferred to a 96-well ViewPlate, which isdesignated as the master plate. The other subculture was transferred toa 96-well ViewPlate, which is designated as the assay plate. The masterplate and the assay plate contain medium so that the clones can becultured. The master pate is continuously cultured under similarconditions.

[0391] The medium in the wells of the assay plate is then removedtherefrom and the clones attached to the well walls are washed twicewith PBS(−). A 5-fold diluted lysis buffer PGC50 (Toyo Ink) is added,respectively, to the clones in the wells of the assay plate at 20μl/well. The assay plate is left standing at room temperature for 30minutes and is set on a luminometer LB96P (Berthold), which is equippedwith an automatic substrate injector. Subsequently, 50 μl of thesubstrate solution PGL100 (Toyo Ink) is automatically dispensed to thelysed clones in the assay plate to measure the luciferase activitytherein with the luminometer LB96P. A plurality of clones, whichexhibited a high luciferase activity are selected therefrom.

[0392] Samples of the selected clones are then cultured at 37° C. for 1to 2 weeks in the presence of 5% CO₂ using dishes having a diameter ofabout 10 cm (Falcon) in charcoal dextran FBS/E-MEM medium.

[0393] The plasmid pRc/RSV-hPPAR γ Kozak is then introduced to thesamples of the selected clones by a lipofection method usinglipofectamine (Life Technologies) to provide a second round of clones.According with the manual provided with the lipofectamine, theconditions under the lipofection method included 5 hours of treatment, 7μg/dish of pRc/RSV-hPPAR γ Kozak and 21 μl/dish of lipofectamine. A DMSOsolution containing 15d prostaglandin J2, which is the natural cognateligand of a human normal PPAR γ, is then added to the resulting secondclones so that the concentration of 15d prostaglandin J2 in the mediumis 10 nM. After culturing the second clones for 2 days, the luciferaseactivity is measured, similarly to the above, for each of the secondclones. The clone in the master plate, which provided the second cloneexhibiting the highest induction of luciferase activity, is selected asthe stably transformed DR1 cassette cell.

[0394] In this regard, the stably transformed DR1 cassette cell can beused in reporter assays with PPAR, RAR, retinoin X receptor, HNF-4,TR-2, TR-4 and the like.

7. Sequence Free Text

[0395] SEQ ID: 1 human normal ERα

[0396] SEQ ID: 2 human mutant ERαK303R

[0397] SEQ ID: 3 human mutant ERαS309F

[0398] SEQ ID: 4 human mutant ERαG390D

[0399] SEQ ID: 5 human mutant ERαM396V

[0400] SEQ ID: 6 human mutant ERαG415V

[0401] SEQ ID: 7 human mutant ERαG494V

[0402] SEQ ID: 8 human mutant ERαK531E

[0403] SEQ ID: 9 human mutant ERαS578P

[0404] SEQ ID: 10 human mutant ERαG390D/S578P

[0405] SEQ ID: 11 Designed oligonucleotide primer for PCR

[0406] SEQ ID: 12 Designed oligonucleotide primer for PCR

[0407] SEQ ID: 13 Designed oligonucleotide for mutagenesis

[0408] SEQ ID: 14 Designed oligonucleotide for mutagenesis

[0409] SEQ ID: 15 Designed oligonucleotide for mutagenesis

[0410] SEQ ID: 16 Designed oligonucleotide for mutagenesis

[0411] SEQ ID: 17 Designed oligonucleotide for mutagenesis

[0412] SEQ ID: 18 Designed oligonucleotide for mutagenesis

[0413] SEQ ID: 19 Designed oligonucleotide for mutagenesis

[0414] SEQ ID :20 Designed oligonucleotide for mutagenesis

[0415] SEQ ID: 21 Designed oligonucleotide for mutagenesis

[0416] SEQ ID: 22 Designed oligonucleotide for mutagenesis

[0417] SEQ ID: 23 Designed oligonucleotide for mutagenesis

[0418] SEQ ID: 24 Designed oligonucleotide for mutagenesis

[0419] SEQ ID: 25 Designed oligonucleotide for mutagenesis

[0420] SEQ ID: 26 Designed oligonucleotide for mutagenesis

[0421] SEQ ID: 27 Designed oligonucleotide for mutagenesis

[0422] SEQ ID: 28 Designed oligonucleotide for mutagenesis

[0423] SEQ ID: 29 Designed oligonucleotide primer for PCR

[0424] SEQ ID: 30 Designed oligonucleotide primer for PCR

[0425] SEQ ID: 31 Designed oligonucleotide primer for PCR

[0426] SEQ ID: 32 Designed oligonucleotide primer for PCR

[0427] SEQ ID: 33 Designed oligonucleotide primer for PCR

[0428] SEQ ID: 34 Designed oligonucleotide primer for PCR

[0429] SEQ ID: 35 Designed oligonucleotide primer for PCR

[0430] SEQ ID: 36 Designed oligonucleotide primer for PCR

[0431] SEQ ID: 37 Designed oligonucleotide primer for PCR

[0432] SEQ ID: 38 Designed oligonucleotide primer for PCR

[0433] SEQ ID: 39 Designed oligonucleotide primer for PCR

[0434] SEQ ID: 40 Designed oligonucleotide primer for PCR

[0435] SEQ ID: 41 Designed oligonucleotide primer for PCR

[0436] SEQ ID: 42 Designed oligonucleotide primer for PCR

[0437] SEQ ID: 43 Designed oligonucleotide primer for PCR

[0438] SEQ ID: 44 Designed oligonucleotide primer for PCR

[0439] SEQ ID: 45 Designed oligonucleotide primer for PCR

[0440] SEQ ID: 46 Designed oligonucleotide primer for PCR

[0441] SEQ ID: 47 Designed oligonucleotide primer for PCR

[0442] SEQ ID: 48 Designed oligonucleotide primer for PCR

[0443] SEQ ID: 49 Designed oligonucleotide primer for PCR

[0444] SEQ ID: 50 Designed oligonucleotide primer for PCR

[0445] SEQ ID: 51 Designed oligonucleotide primer for PCR

[0446] SEQ ID: 52 Designed oligonucleotide primer for PCR

[0447] SEQ ID: 53 Designed oligonucleotide primer for PCR

[0448] SEQ ID: 54 Designed oligonucleotide primer for PCR

[0449] SEQ ID: 55 Designed oligonucleotide primer for PCR

[0450] SEQ ID: 56 Designed oligonucleotide primer for PCR

[0451] SEQ ID: 57 Designed oligonucleotide primer for PCR

[0452] SEQ ID: 58 Designed oligonucleotide primer for PCR

[0453] SEQ ID: 59 Designed oligonucleotide primer for PCR

[0454] SEQ ID: 60 Designed oligonucleotide primer for PCR

[0455] SEQ ID: 61 Designed oligonucleotide primer for PCR

[0456] SEQ ID: 62 Designed oligonucleotide primer for PCR

[0457] SEQ ID: 63 Designed oligonucleotide primer for PCR

[0458] SEQ ID: 64 Designed oligonucleotide primer for PCR

[0459] SEQ ID: 65 Designed oligonucleotide primer for PCR

[0460] SEQ ID: 66 Designed oligonucleotide primer for PCR

[0461] SEQ ID: 67 Designed oligonucleotide primer for PCR

[0462] SEQ ID: 68 Designed oligonucleotide primer for PCR

[0463] SEQ ID: 69 Designed oligonucleotide primer for PCR

[0464] SEQ ID: 70 Designed oligonucleotide primer for PCR

[0465] SEQ ID: 71 Designed oligonucleotide primer for PCR

[0466] SEQ ID: 72 Designed oligonucleotide primer for PCR

[0467] SEQ ID: 73 Designed oligonucleotide primer for PCR

[0468] SEQ ID: 74 Designed oligonucleotide primer for PCR

[0469] SEQ ID: 75 Designed oligonucleotide primer for PCR

[0470] SEQ ID: 76 Designed oligonucleotide primer for PCR

[0471] SEQ ID: 77 Designed oligonucleotide primer for PCR

[0472] SEQ ID: 78 Designed oligonucleotide primer for PCR

[0473] SEQ ID: 79 Designed oligonucleotide primer for PCR

[0474] SEQ ID: 80 Designed oligonucleotide primer for PCR

[0475] SEQ ID: 81 Designed oligonucleotide primer for PCR

[0476] SEQ ID: 82 Designed oligonucleotide primer for PCR

[0477] SEQ ID: 83 Designed oligonucleotide primer for PCR

[0478] SEQ ID: 84 Designed oligonucleotide primer for PCR

[0479] SEQ ID: 85 Designed oligonucleotide primer for PCR

[0480] SEQ ID: 86 Designed oligonucleotide primer for PCR

[0481] SEQ ID: 87 Designed oligonucleotide primer for PCR

[0482] SEQ ID: 88 Designed oligonucleotide primer for PCR

[0483] SEQ ID: 89 Designed oligonucleotide primer for PCR

[0484] SEQ ID: 90 Designed oligonucleotide primer for PCR

[0485] SEQ ID: 91 Designed oligonucleotide primer for PCR

[0486] SEQ ID: 92 Designed oligonucleotide primer for PCR

[0487] SEQ ID: 93 Designed oligonucleotide primer for PCR

[0488] SEQ ID: 94 Designed oligonucleotide primer for PCR

[0489] SEQ ID: 95 Designed oligonucleotide primer for PCR

[0490] SEQ ID: 96 Designed oligonucleotide primer for PCR

[0491] SEQ ID: 97 Designed oligonucleotide primer for PCR

[0492] SEQ ID: 98 Designed oligonucleotide primer for PCR

[0493] SEQ ID: 99 Designed oligonucleotide primer for PCR

[0494] SEQ ID: 100 Designed oligonucleotide primer for PCR

[0495] SEQ ID: 101 Designed oligonucleotide primer for PCR

[0496] SEQ ID: 102 Designed oligonucleotide primer for PCR

[0497] SEQ ID: 103 Designed oligonucleotide primer for PCR

[0498] SEQ ID: 104 Designed oligonucleotide primer for PCR

[0499] SEQ ID: 105 Designed oligonucleotide primer for PCR

[0500] SEQ ID: 106 Designed oligonucleotide primer for PCR

[0501] SEQ ID: 107 Designed oligonucleotide primer for PCR

[0502] SEQ ID: 108 Designed oligonucleotide primer for PCR

[0503] SEQ ID: 109 Designed oligonucleotide primer for PCR

[0504] SEQ ID: 110 Designed oligonucleotide primer for PCR

[0505] SEQ ID: 111 Designed oligonucleotide probe for Southernhybridization

[0506] SEQ ID: 112 Designed oligonucleotide probe for Southernhybridization

[0507] SEQ ID: 113 Designed oligonucleotide probe for Southernhybridization

[0508] SEQ ID: 114 Designed oligonucleotide probe for Southernhybridization

[0509] SEQ ID: 115 Designed oligonucleotide probe for Southernhybridization

[0510] SEQ ID: 116 Designed oligonucleotide probe for Southernhybridization

[0511] SEQ ID: 117 Designed oligonucleotide probe for Southernhybridization

[0512] SEQ ID: 118 Designed oligonucleotide probe for Southernhybridization

[0513] SEQ ID: 119 Designed oligonucleotide probe for Southernhybridization

[0514] SEQ ID: 120 Designed oligonucleotide probe for Southernhybridization

[0515] SEQ ID: 121 Designed oligonucleotide probe for Southernhybridization

[0516] SEQ ID: 122 Designed oligonucleotide probe for Southernhybridization

[0517] SEQ ID: 123 Designed oligonucleotide probe for Southernhybridization

[0518] SEQ ID: 124 Designed oligonucleotide probe for Southernhybridization

[0519] SEQ ID: 125 Designed oligonucleotide probe for Southernhybridization

[0520] SEQ ID: 126 Designed oligonucleotide probe for Southernhybridization

[0521] SEQ ID: 127 Designed oligonucleotide probe for Southernhybridization

[0522] SEQ ID: 128 Designed oligonucleotide probe for Southernhybridization

[0523] SEQ ID: 129 Designed oligonucleotide probe for Southernhybridization

[0524] SEQ ID: 130 Designed oligonucleotide probe for Southernhybridization

[0525] SEQ ID: 131 Designed oligonucleotide probe for Southernhybridization.

[0526] SEQ ID: 132 Designed oligonucleotide probe for Southernhybridization

[0527] SEQ ID: 133 Designed oligonucleotide probe for Southernhybridization

[0528] SEQ ID: 134 Designed oligonucleotide probe for Southernhybridization

[0529] SEQ ID: 135 Designed oligonucleotide probe for Southernhybridization

[0530] SEQ ID: 136 Designed oligonucleotide probe for Southernhybridization

[0531] SEQ ID: 137 Designed oligonucleotide probe for Southernhybridization

[0532] SEQ ID: 138 Designed oligonucleotide probe for Southernhybridization

[0533] SEQ ID: 139 Designed oligonucleotide probe for Southernhybridization

[0534] SEQ ID: 140 Designed oligonucleotide probe for Southernhybridization

[0535] SEQ ID: 141 Designed oligonucleotide probe for Southernhybridization

[0536] SEQ ID: 142 Designed oligonucleotide probe for Southernhybridization

[0537] SEQ ID: 143 Designed oligonucleotide probe for Southernhybridization

[0538] SEQ ID: 144 Designed oligonucleotide probe for Southernhybridization

[0539] SEQ ID: 145 Designed oligonucleotide probe for Southernhybridization

[0540] SEQ ID: 146 Designed oligonucleotide probe for Southernhybridization

[0541] SEQ ID: 147 Designed oligonucleotide probe for Southernhybridization

[0542] SEQ ID: 148 Designed oligonucleotide probe for Southernhybridization

[0543] SEQ ID: 149 Designed oligonucleotide probe for Southernhybridization

[0544] SEQ ID: 150 Designed oligonucleotide probe for Southernhybridization

[0545] SEQ ID: 151 Designed oligonucleotide primer for PCR

[0546] SEQ ID: 152 Designed oligonucleotide for mutagenesis

[0547] SEQ ID: 153 Designed oligonucleotide for mutagenesis

[0548] SEQ ID: 154 Designed oligonucleotide for mutagenesis

[0549] SEQ ID: 155 Designed oligonucleotide for mutagenesis

[0550] SEQ ID: 156 Designed oligonucleotide for mutagenesis

[0551] SEQ ID: 157 Designed oligonucleotide for mutagenesis

[0552] SEQ ID: 158 Designed oligonucleotide primer for PCR

[0553] SEQ ID: 159 Designed oligonucleotide primer for PCR

[0554] SEQ ID: 160 Designed oligonucleotide primer for PCR

[0555] SEQ ID: 161 Designed oligonucleotide for synthesis

[0556] SEQ ID: 162 Designed oligonucleotide for synthesis

[0557] SEQ ID: 163 Designed oligonucleotide for synthesis

[0558] SEQ ID: 164 Designed oligonucleotide primer for PCR

[0559] SEQ ID: 165 Designed oligonucleotide primer for PCR

[0560] SEQ ID: 166 Designed oligonucleotide primer for PCR

[0561] SEQ ID: 167 Designed oligonucleotide primer for PCR

[0562] SEQ ID: 168 Designed oligonucleotide primer for PCR

[0563] SEQ ID: 169 Designed oligonucleotide primer for PCR

[0564] SEQ ID: 170 Designed oligonucleotide primer for PCR

[0565] SEQ ID: 171 Designed oligonucleotide primer for PCR

[0566] SEQ ID: 172 Designed oligonucleotide primer for PCR

[0567] SEQ ID: 173 Designed oligonucleotide primer for PCR

[0568] SEQ ID: 174 Designed oligonucleotide primer for PCR

[0569] SEQ ID: 175 Designed oligonucleotide primer for PCR

[0570] SEQ ID: 176 Designed oligonucleotide primer for PCR

[0571] SEQ ID: 177 Designed oligonucleotide primer for PCR

[0572] SEQ ID: 178 Designed oligonucleotide primer for PCR

[0573] SEQ ID: 179 Designed oligonucleotide primer for PCR

[0574] SEQ ID: 180 Designed oligonucleotide primer for PCR

[0575] SEQ ID: 181 Designed oligonucleotide primer for PCR

[0576] SEQ ID: 182 Designed oligonucleotide primer for PCR

[0577] SEQ ID: 183 Designed oligonucleotide primer for PCR

[0578] SEQ ID: 184 Designed oligonucleotide primer for PCR

[0579] SEQ ID: 185 Designed oligonucleotide primer for PCR

[0580] SEQ ID: 186 Designed oligonucleotide primer for PCR

[0581] SEQ ID: 187 Designed oligonucleotide primer for PCR

[0582] SEQ ID: 188 Designed oligonucleotide primer for PCR

[0583] SEQ ID: 189 Designed oligonucleotide primer for PCR

[0584] SEQ ID: 190 Designed oligonucleotide primer for PCR

[0585] SEQ ID: 191 Designed oligonucleotide primer for PCR

[0586] SEQ ID: 192 Designed oligonucleotide primer for PCR

[0587] SEQ ID: 193 Designed oligonucleotide primer for PCR

[0588] SEQ ID: 194 Designed oligonucleotide primer for PCR

[0589] SEQ ID: 195 Designed oligonucleotide primer for PCR

[0590] SEQ ID: 196 Designed oligonucleotide primer for PCR

[0591] SEQ ID: 197 Designed oligonucleotide primer for PCR

[0592] SEQ ID: 198 Designed oligonucleotide primer for PCR

[0593] SEQ ID: 199 Designed oligonucleotide primer for PCR

[0594] SEQ ID: 200 Designed oligonucleotide primer for PCR

[0595] SEQ ID: 201 Designed oligonucleotide primer for PCR

[0596] SEQ ID: 202 Designed oligonucleotide primer for PCR

[0597] SEQ ID: 203 Designed oligonucleotide primer for PCR

[0598] SEQ ID: 204 Designed oligonucleotide for synthesis

[0599] SEQ ID: 205 Designed oligonucleotide primer for PCR

[0600] SEQ ID: 206 Designed oligonucleotide primer for PCR

[0601] SEQ ID: 207 Designed oligonucleotide primer for PCR

[0602] SEQ ID: 208 Designed oligonucleotide primer for PCR

[0603] SEQ ID: 209 Designed oligonucleotide for synthesis

[0604] SEQ ID: 210 Designed oligonucleotide primer for PCR

[0605] SEQ ID: 211 Designed oligonucleotide primer for PCR

[0606] SEQ ID: 212 Designed oligonucleotide primer for PCR

[0607] SEQ ID: 213 designed oligonucleotide for synthesis

1 213 1 595 PRT Homo sapiens 1 Met Thr Met Thr Leu His Thr Lys Ala SerGly Met Ala Leu Leu His 1 5 10 15 Gln Ile Gln Gly Asn Glu Leu Glu ProLeu Asn Arg Pro Gln Leu Lys 20 25 30 Ile Pro Leu Glu Arg Pro Leu Gly GluVal Tyr Leu Asp Ser Ser Lys 35 40 45 Pro Ala Val Tyr Asn Tyr Pro Glu GlyAla Ala Tyr Glu Phe Asn Ala 50 55 60 Ala Ala Ala Ala Asn Ala Gln Val TyrGly Gln Thr Gly Leu Pro Tyr 65 70 75 80 Gly Pro Gly Ser Glu Ala Ala AlaPhe Gly Ser Asn Gly Leu Gly Gly 85 90 95 Phe Pro Pro Leu Asn Ser Val SerPro Ser Pro Leu Met Leu Leu His 100 105 110 Pro Pro Pro Gln Leu Ser ProPhe Leu Gln Pro His Gly Gln Gln Val 115 120 125 Pro Tyr Tyr Leu Glu AsnGlu Pro Ser Gly Tyr Thr Val Arg Glu Ala 130 135 140 Gly Pro Pro Ala PheTyr Arg Pro Asn Ser Asp Asn Arg Arg Gln Gly 145 150 155 160 Gly Arg GluArg Leu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met 165 170 175 Glu SerAla Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 180 185 190 SerGly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 205Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr 210 215220 Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys 225230 235 240 Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly IleArg 245 250 255 Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg GlnArg Asp 260 265 270 Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly AspMet Arg Ala 275 280 285 Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys ArgSer Lys Lys Asn 290 295 300 Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln MetVal Ser Ala Leu Leu 305 310 315 320 Asp Ala Glu Pro Pro Ile Leu Tyr SerGlu Tyr Asp Pro Thr Arg Pro 325 330 335 Phe Ser Glu Ala Ser Met Met GlyLeu Leu Thr Asn Leu Ala Asp Arg 340 345 350 Glu Leu Val His Met Ile AsnTrp Ala Lys Arg Val Pro Gly Phe Val 355 360 365 Asp Leu Thr Leu His AspGln Val His Leu Leu Glu Cys Ala Trp Leu 370 375 380 Glu Ile Leu Met IleGly Leu Val Trp Arg Ser Met Glu His Pro Gly 385 390 395 400 Lys Leu LeuPhe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 405 410 415 Cys ValGlu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 420 425 430 SerArg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu 435 440 445Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 450 455460 Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp 465470 475 480 Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly LeuThr 485 490 495 Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu IleLeu Ser 500 505 510 His Ile Arg His Met Ser Asn Lys Gly Met Glu His LeuTyr Ser Met 515 520 525 Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu LeuLeu Glu Met Leu 530 535 540 Asp Ala His Arg Leu His Ala Pro Thr Ser ArgGly Gly Ala Ser Val 545 550 555 560 Glu Glu Thr Asp Gln Ser His Leu AlaThr Ala Gly Ser Thr Ser Ser 565 570 575 His Pro Leu Gln Lys Tyr Tyr IleThr Gly Glu Ala Glu Gly Phe Pro 580 585 590 Ala Thr Val 595 2 595 PRTHomo sapiens 2 Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala LeuLeu His 1 5 10 15 Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg ProGln Leu Lys 20 25 30 Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu AspSer Ser Lys 35 40 45 Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr GluPhe Asn Ala 50 55 60 Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr GlyLeu Pro Tyr 65 70 75 80 Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser AsnGly Leu Gly Gly 85 90 95 Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro LeuMet Leu Leu His 100 105 110 Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln ProHis Gly Gln Gln Val 115 120 125 Pro Tyr Tyr Leu Glu Asn Glu Pro Ser GlyTyr Thr Val Arg Glu Ala 130 135 140 Gly Pro Pro Ala Phe Tyr Arg Pro AsnSer Asp Asn Arg Arg Gln Gly 145 150 155 160 Gly Arg Glu Arg Leu Ala SerThr Asn Asp Lys Gly Ser Met Ala Met 165 170 175 Glu Ser Ala Lys Glu ThrArg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 180 185 190 Ser Gly Tyr His TyrGly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 205 Phe Lys Arg SerIle Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr 210 215 220 Asn Gln CysThr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys 225 230 235 240 ArgLeu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg 245 250 255Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp 260 265270 Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala 275280 285 Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Arg Asn290 295 300 Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala LeuLeu 305 310 315 320 Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp ProThr Arg Pro 325 330 335 Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr AsnLeu Ala Asp Arg 340 345 350 Glu Leu Val His Met Ile Asn Trp Ala Lys ArgVal Pro Gly Phe Val 355 360 365 Asp Leu Thr Leu His Asp Gln Val His LeuLeu Glu Cys Ala Trp Leu 370 375 380 Glu Ile Leu Met Ile Gly Leu Val TrpArg Ser Met Glu His Pro Gly 385 390 395 400 Lys Leu Leu Phe Ala Pro AsnLeu Leu Leu Asp Arg Asn Gln Gly Lys 405 410 415 Cys Val Glu Gly Met ValGlu Ile Phe Asp Met Leu Leu Ala Thr Ser 420 425 430 Ser Arg Phe Arg MetMet Asn Leu Gln Gly Glu Glu Phe Val Cys Leu 435 440 445 Lys Ser Ile IleLeu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 450 455 460 Thr Leu LysSer Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp 465 470 475 480 LysIle Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr 485 490 495Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser 500 505510 His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met 515520 525 Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu530 535 540 Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala SerVal 545 550 555 560 Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly SerThr Ser Ser 565 570 575 His Pro Leu Gln Lys Tyr Tyr Ile Thr Gly Glu AlaGlu Gly Phe Pro 580 585 590 Ala Thr Val 595 3 595 PRT Homo sapiens 3 MetThr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu Leu His 1 5 10 15Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys 20 25 30Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu Asp Ser Ser Lys 35 40 45Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr Gly Leu Pro Tyr 65 70 7580 Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly 85 9095 Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro Leu Met Leu Leu His 100105 110 Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val115 120 125 Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg GluAla 130 135 140 Gly Pro Pro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg ArgGln Gly 145 150 155 160 Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys GlySer Met Ala Met 165 170 175 Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala ValCys Asn Asp Tyr Ala 180 185 190 Ser Gly Tyr His Tyr Gly Val Trp Ser CysGlu Gly Cys Lys Ala Phe 195 200 205 Phe Lys Arg Ser Ile Gln Gly His AsnAsp Tyr Met Cys Pro Ala Thr 210 215 220 Asn Gln Cys Thr Ile Asp Lys AsnArg Arg Lys Ser Cys Gln Ala Cys 225 230 235 240 Arg Leu Arg Lys Cys TyrGlu Val Gly Met Met Lys Gly Gly Ile Arg 245 250 255 Lys Asp Arg Arg GlyGly Arg Met Leu Lys His Lys Arg Gln Arg Asp 260 265 270 Asp Gly Glu GlyArg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala 275 280 285 Ala Asn LeuTrp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn 290 295 300 Ser LeuAla Leu Phe Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu 305 310 315 320Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro 325 330335 Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg 340345 350 Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val355 360 365 Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala TrpLeu 370 375 380 Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu HisPro Gly 385 390 395 400 Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp ArgAsn Gln Gly Lys 405 410 415 Cys Val Glu Gly Met Val Glu Ile Phe Asp MetLeu Leu Ala Thr Ser 420 425 430 Ser Arg Phe Arg Met Met Asn Leu Gln GlyGlu Glu Phe Val Cys Leu 435 440 445 Lys Ser Ile Ile Leu Leu Asn Ser GlyVal Tyr Thr Phe Leu Ser Ser 450 455 460 Thr Leu Lys Ser Leu Glu Glu LysAsp His Ile His Arg Val Leu Asp 465 470 475 480 Lys Ile Thr Asp Thr LeuIle His Leu Met Ala Lys Ala Gly Leu Thr 485 490 495 Leu Gln Gln Gln HisGln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser 500 505 510 His Ile Arg HisMet Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met 515 520 525 Lys Cys LysAsn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu 530 535 540 Asp AlaHis Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val 545 550 555 560Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser 565 570575 His Pro Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro 580585 590 Ala Thr Val 595 4 595 PRT Homo sapiens 4 Met Thr Met Thr Leu HisThr Lys Ala Ser Gly Met Ala Leu Leu His 1 5 10 15 Gln Ile Gln Gly AsnGlu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys 20 25 30 Ile Pro Leu Glu ArgPro Leu Gly Glu Val Tyr Leu Asp Ser Ser Lys 35 40 45 Pro Ala Val Tyr AsnTyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60 Ala Ala Ala Ala AsnAla Gln Val Tyr Gly Gln Thr Gly Leu Pro Tyr 65 70 75 80 Gly Pro Gly SerGlu Ala Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly 85 90 95 Phe Pro Pro LeuAsn Ser Val Ser Pro Ser Pro Leu Met Leu Leu His 100 105 110 Pro Pro ProGln Leu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val 115 120 125 Pro TyrTyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala 130 135 140 GlyPro Pro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg Gln Gly 145 150 155160 Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met 165170 175 Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala180 185 190 Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys AlaPhe 195 200 205 Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys ProAla Thr 210 215 220 Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser CysGln Ala Cys 225 230 235 240 Arg Leu Arg Lys Cys Tyr Glu Val Gly Met MetLys Gly Gly Ile Arg 245 250 255 Lys Asp Arg Arg Gly Gly Arg Met Leu LysHis Lys Arg Gln Arg Asp 260 265 270 Asp Gly Glu Gly Arg Gly Glu Val GlySer Ala Gly Asp Met Arg Ala 275 280 285 Ala Asn Leu Trp Pro Ser Pro LeuMet Ile Lys Arg Ser Lys Lys Asn 290 295 300 Ser Leu Ala Leu Ser Leu ThrAla Asp Gln Met Val Ser Ala Leu Leu 305 310 315 320 Asp Ala Glu Pro ProIle Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro 325 330 335 Phe Ser Glu AlaSer Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg 340 345 350 Glu Leu ValHis Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val 355 360 365 Asp LeuThr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu 370 375 380 GluIle Leu Met Ile Asp Leu Val Trp Arg Ser Met Glu His Pro Gly 385 390 395400 Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 405410 415 Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser420 425 430 Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val CysLeu 435 440 445 Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe LeuSer Ser 450 455 460 Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His ArgVal Leu Asp 465 470 475 480 Lys Ile Thr Asp Thr Leu Ile His Leu Met AlaLys Ala Gly Leu Thr 485 490 495 Leu Gln Gln Gln His Gln Arg Leu Ala GlnLeu Leu Leu Ile Leu Ser 500 505 510 His Ile Arg His Met Ser Asn Lys GlyMet Glu His Leu Tyr Ser Met 515 520 525 Lys Cys Lys Asn Val Val Pro LeuTyr Asp Leu Leu Leu Glu Met Leu 530 535 540 Asp Ala His Arg Leu His AlaPro Thr Ser Arg Gly Gly Ala Ser Val 545 550 555 560 Glu Glu Thr Asp GlnSer His Leu Ala Thr Ala Gly Ser Thr Ser Ser 565 570 575 His Ser Leu GlnLys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro 580 585 590 Ala Thr Val595 5 595 PRT Homo sapiens 5 Met Thr Met Thr Leu His Thr Lys Ala Ser GlyMet Ala Leu Leu His 1 5 10 15 Gln Ile Gln Gly Asn Glu Leu Glu Pro LeuAsn Arg Pro Gln Leu Lys 20 25 30 Ile Pro Leu Glu Arg Pro Leu Gly Glu ValTyr Leu Asp Ser Ser Lys 35 40 45 Pro Ala Val Tyr Asn Tyr Pro Glu Gly AlaAla Tyr Glu Phe Asn Ala 50 55 60 Ala Ala Ala Ala Asn Ala Gln Val Tyr GlyGln Thr Gly Leu Pro Tyr 65 70 75 80 Gly Pro Gly Ser Glu Ala Ala Ala PheGly Ser Asn Gly Leu Gly Gly 85 90 95 Phe Pro Pro Leu Asn Ser Val Ser ProSer Pro Leu Met Leu Leu His 100 105 110 Pro Pro Pro Gln Leu Ser Pro PheLeu Gln Pro His Gly Gln Gln Val 115 120 125 Pro Tyr Tyr Leu Glu Asn GluPro Ser Gly Tyr Thr Val Arg Glu Ala 130 135 140 Gly Pro Pro Ala Phe TyrArg Pro Asn Ser Asp Asn Arg Arg Gln Gly 145 150 155 160 Gly Arg Glu ArgLeu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met 165 170 175 Glu Ser AlaLys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 180 185 190 Ser GlyTyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 205 PheLys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr 210 215 220Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys 225 230235 240 Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg245 250 255 Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln ArgAsp 260 265 270 Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp MetArg Ala 275 280 285 Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg SerLys Lys Asn 290 295 300 Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met ValSer Ala Leu Leu 305 310 315 320 Asp Ala Glu Pro Pro Ile Leu Tyr Ser GluTyr Asp Pro Thr Arg Pro 325 330 335 Phe Ser Glu Ala Ser Met Met Gly LeuLeu Thr Asn Leu Ala Asp Arg 340 345 350 Glu Leu Val His Met Ile Asn TrpAla Lys Arg Val Pro Gly Phe Val 355 360 365 Asp Leu Thr Leu His Asp GlnVal His Leu Leu Glu Cys Ala Trp Leu 370 375 380 Glu Ile Leu Met Ile GlyLeu Val Trp Arg Ser Val Glu His Pro Gly 385 390 395 400 Lys Leu Leu PheAla Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 405 410 415 Cys Val GluGly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 420 425 430 Ser ArgPhe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu 435 440 445 LysSer Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 450 455 460Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp 465 470475 480 Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr485 490 495 Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile LeuSer 500 505 510 His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu TyrSer Met 515 520 525 Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu LeuGlu Met Leu 530 535 540 Asp Ala His Arg Leu His Ala Pro Thr Ser Arg GlyGly Ala Ser Val 545 550 555 560 Glu Glu Thr Asp Gln Ser His Leu Ala ThrAla Gly Ser Thr Ser Ser 565 570 575 His Pro Leu Gln Lys Tyr Tyr Ile ThrGly Glu Ala Glu Gly Phe Pro 580 585 590 Ala Thr Val 595 6 595 PRT Homosapiens 6 Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu LeuHis 1 5 10 15 Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro GlnLeu Lys 20 25 30 Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu Asp SerSer Lys 35 40 45 Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Glu PheAsn Ala 50 55 60 Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr Gly LeuPro Tyr 65 70 75 80 Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn GlyLeu Gly Gly 85 90 95 Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro Leu MetLeu Leu His 100 105 110 Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln Pro HisGly Gln Gln Val 115 120 125 Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly TyrThr Val Arg Glu Ala 130 135 140 Gly Pro Pro Ala Phe Tyr Arg Pro Asn SerAsp Asn Arg Arg Gln Gly 145 150 155 160 Gly Arg Glu Arg Leu Ala Ser ThrAsn Asp Lys Gly Ser Met Ala Met 165 170 175 Glu Ser Ala Lys Glu Thr ArgTyr Cys Ala Val Cys Asn Asp Tyr Ala 180 185 190 Ser Gly Tyr His Tyr GlyVal Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 205 Phe Lys Arg Ser IleGln Gly His Asn Asp Tyr Met Cys Pro Ala Thr 210 215 220 Asn Gln Cys ThrIle Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys 225 230 235 240 Arg LeuArg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg 245 250 255 LysAsp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp 260 265 270Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala 275 280285 Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn 290295 300 Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu305 310 315 320 Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro ThrArg Pro 325 330 335 Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn LeuAla Asp Arg 340 345 350 Glu Leu Val His Met Ile Asn Trp Ala Lys Arg ValPro Gly Phe Val 355 360 365 Asp Leu Thr Leu His Asp Gln Val His Leu LeuGlu Cys Ala Trp Leu 370 375 380 Glu Ile Leu Met Ile Gly Leu Val Trp ArgSer Met Glu His Pro Gly 385 390 395 400 Lys Leu Leu Phe Ala Pro Asn LeuLeu Leu Asp Arg Asn Gln Val Lys 405 410 415 Cys Val Glu Gly Met Val GluIle Phe Asp Met Leu Leu Ala Thr Ser 420 425 430 Ser Arg Phe Arg Met MetAsn Leu Gln Gly Glu Glu Phe Val Cys Leu 435 440 445 Lys Ser Ile Ile LeuLeu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 450 455 460 Thr Leu Lys SerLeu Glu Glu Lys Asp His Ile His Arg Val Leu Asp 465 470 475 480 Lys IleThr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr 485 490 495 LeuGln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser 500 505 510His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met 515 520525 Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu 530535 540 Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val545 550 555 560 Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser ThrSer Ser 565 570 575 His Pro Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala GluGly Phe Pro 580 585 590 Ala Thr Val 595 7 595 PRT Homo sapiens 7 Met ThrMet Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu Leu His 1 5 10 15 GlnIle Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys 20 25 30 IlePro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu Asp Ser Ser Lys 35 40 45 ProAla Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60 AlaAla Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr Gly Leu Pro Tyr 65 70 75 80Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly 85 90 95Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro Leu Met Leu Leu His 100 105110 Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val 115120 125 Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala130 135 140 Gly Pro Pro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg GlnGly 145 150 155 160 Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly SerMet Ala Met 165 170 175 Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val CysAsn Asp Tyr Ala 180 185 190 Ser Gly Tyr His Tyr Gly Val Trp Ser Cys GluGly Cys Lys Ala Phe 195 200 205 Phe Lys Arg Ser Ile Gln Gly His Asn AspTyr Met Cys Pro Ala Thr 210 215 220 Asn Gln Cys Thr Ile Asp Lys Asn ArgArg Lys Ser Cys Gln Ala Cys 225 230 235 240 Arg Leu Arg Lys Cys Tyr GluVal Gly Met Met Lys Gly Gly Ile Arg 245 250 255 Lys Asp Arg Arg Gly GlyArg Met Leu Lys His Lys Arg Gln Arg Asp 260 265 270 Asp Gly Glu Gly ArgGly Glu Val Gly Ser Ala Gly Asp Met Arg Ala 275 280 285 Ala Asn Leu TrpPro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn 290 295 300 Ser Leu AlaLeu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu 305 310 315 320 AspAla Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro 325 330 335Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg 340 345350 Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val 355360 365 Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu370 375 380 Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His ProGly 385 390 395 400 Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg AsnGln Gly Lys 405 410 415 Cys Val Glu Gly Met Val Glu Ile Phe Asp Met LeuLeu Ala Thr Ser 420 425 430 Ser Arg Phe Arg Met Met Asn Leu Gln Gly GluGlu Phe Val Cys Leu 435 440 445 Lys Ser Ile Ile Leu Leu Asn Ser Gly ValTyr Thr Phe Leu Ser Ser 450 455 460 Thr Leu Lys Ser Leu Glu Glu Lys AspHis Ile His Arg Val Leu Asp 465 470 475 480 Lys Ile Thr Asp Thr Leu IleHis Leu Met Ala Lys Ala Val Leu Thr 485 490 495 Leu Gln Gln Gln His GlnArg Leu Ala Gln Leu Leu Leu Ile Leu Ser 500 505 510 His Ile Arg His MetSer Asn Lys Gly Met Glu His Leu Tyr Ser Met 515 520 525 Lys Cys Lys AsnVal Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu 530 535 540 Asp Ala HisArg Leu His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val 545 550 555 560 GluGlu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser 565 570 575His Pro Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro 580 585590 Ala Thr Val 595 8 595 PRT Homo sapiens 8 Met Thr Met Thr Leu His ThrLys Ala Ser Gly Met Ala Leu Leu His 1 5 10 15 Gln Ile Gln Gly Asn GluLeu Glu Pro Leu Asn Arg Pro Gln Leu Lys 20 25 30 Ile Pro Leu Glu Arg ProLeu Gly Glu Val Tyr Leu Asp Ser Ser Lys 35 40 45 Pro Ala Val Tyr Asn TyrPro Glu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60 Ala Ala Ala Ala Asn AlaGln Val Tyr Gly Gln Thr Gly Leu Pro Tyr 65 70 75 80 Gly Pro Gly Ser GluAla Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly 85 90 95 Phe Pro Pro Leu AsnSer Val Ser Pro Ser Pro Leu Met Leu Leu His 100 105 110 Pro Pro Pro GlnLeu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val 115 120 125 Pro Tyr TyrLeu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala 130 135 140 Gly ProPro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg Gln Gly 145 150 155 160Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met 165 170175 Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 180185 190 Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe195 200 205 Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro AlaThr 210 215 220 Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys GlnAla Cys 225 230 235 240 Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met LysGly Gly Ile Arg 245 250 255 Lys Asp Arg Arg Gly Gly Arg Met Leu Lys HisLys Arg Gln Arg Asp 260 265 270 Asp Gly Glu Gly Arg Gly Glu Val Gly SerAla Gly Asp Met Arg Ala 275 280 285 Ala Asn Leu Trp Pro Ser Pro Leu MetIle Lys Arg Ser Lys Lys Asn 290 295 300 Ser Leu Ala Leu Ser Leu Thr AlaAsp Gln Met Val Ser Ala Leu Leu 305 310 315 320 Asp Ala Glu Pro Pro IleLeu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro 325 330 335 Phe Ser Glu Ala SerMet Met Gly Leu Leu Thr Asn Leu Ala Asp Arg 340 345 350 Glu Leu Val HisMet Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val 355 360 365 Asp Leu ThrLeu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu 370 375 380 Glu IleLeu Met Ile Gly Leu Val Trp Arg Ser Val Glu His Pro Gly 385 390 395 400Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 405 410415 Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 420425 430 Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu435 440 445 Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu SerSer 450 455 460 Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg ValLeu Asp 465 470 475 480 Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala LysAla Gly Leu Thr 485 490 495 Leu Gln Gln Gln His Gln Arg Leu Ala Gln LeuLeu Leu Ile Leu Ser 500 505 510 His Ile Arg His Met Ser Asn Lys Gly MetGlu His Leu Tyr Ser Met 515 520 525 Lys Cys Glu Asn Val Val Pro Leu TyrAsp Leu Leu Leu Glu Met Leu 530 535 540 Asp Ala His Arg Leu His Ala ProThr Ser Arg Gly Gly Ala Ser Val 545 550 555 560 Glu Glu Thr Asp Gln SerHis Leu Ala Thr Ala Gly Ser Thr Ser Ser 565 570 575 His Ser Leu Gln LysTyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro 580 585 590 Ala Thr Val 5959 595 PRT Homo sapiens 9 Met Thr Met Thr Leu His Thr Lys Ala Ser Gly MetAla Leu Leu His 1 5 10 15 Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu AsnArg Pro Gln Leu Lys 20 25 30 Ile Pro Leu Glu Arg Pro Leu Gly Glu Val TyrLeu Asp Ser Ser Lys 35 40 45 Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala AlaTyr Glu Phe Asn Ala 50 55 60 Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly GlnThr Gly Leu Pro Tyr 65 70 75 80 Gly Pro Gly Ser Glu Ala Ala Ala Phe GlySer Asn Gly Leu Gly Gly 85 90 95 Phe Pro Pro Leu Asn Ser Val Ser Pro SerPro Leu Met Leu Leu His 100 105 110 Pro Pro Pro Gln Leu Ser Pro Phe LeuGln Pro His Gly Gln Gln Val 115 120 125 Pro Tyr Tyr Leu Glu Asn Glu ProSer Gly Tyr Thr Val Arg Glu Ala 130 135 140 Gly Pro Pro Ala Phe Tyr ArgPro Asn Ser Asp Asn Arg Arg Gln Gly 145 150 155 160 Gly Arg Glu Arg LeuAla Ser Thr Asn Asp Lys Gly Ser Met Ala Met 165 170 175 Glu Ser Ala LysGlu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 180 185 190 Ser Gly TyrHis Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 205 Phe LysArg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr 210 215 220 AsnGln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys 225 230 235240 Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg 245250 255 Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp260 265 270 Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met ArgAla 275 280 285 Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser LysLys Asn 290 295 300 Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val SerAla Leu Leu 305 310 315 320 Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu TyrAsp Pro Thr Arg Pro 325 330 335 Phe Ser Glu Ala Ser Met Met Gly Leu LeuThr Asn Leu Ala Asp Arg 340 345 350 Glu Leu Val His Met Ile Asn Trp AlaLys Arg Val Pro Gly Phe Val 355 360 365 Asp Leu Thr Leu His Asp Gln ValHis Leu Leu Glu Cys Ala Trp Leu 370 375 380 Glu Ile Leu Met Ile Gly LeuVal Trp Arg Ser Met Glu His Pro Gly 385 390 395 400 Lys Leu Leu Phe AlaPro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 405 410 415 Cys Val Glu GlyMet Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 420 425 430 Ser Arg PheArg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu 435 440 445 Lys SerIle Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 450 455 460 ThrLeu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp 465 470 475480 Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr 485490 495 Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser500 505 510 His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr SerMet 515 520 525 Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu GluMet Leu 530 535 540 Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly GlyAla Ser Val 545 550 555 560 Glu Glu Thr Asp Gln Ser His Leu Ala Thr AlaGly Ser Thr Ser Ser 565 570 575 His Pro Leu Gln Lys Tyr Tyr Ile Thr GlyGlu Ala Glu Gly Phe Pro 580 585 590 Ala Thr Val 595 10 595 PRT Homosapiens 10 Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu LeuHis 1 5 10 15 Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro GlnLeu Lys 20 25 30 Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu Asp SerSer Lys 35 40 45 Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Glu PheAsn Ala 50 55 60 Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr Gly LeuPro Tyr 65 70 75 80 Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn GlyLeu Gly Gly 85 90 95 Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro Leu MetLeu Leu His 100 105 110 Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln Pro HisGly Gln Gln Val 115 120 125 Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly TyrThr Val Arg Glu Ala 130 135 140 Gly Pro Pro Ala Phe Tyr Arg Pro Asn SerAsp Asn Arg Arg Gln Gly 145 150 155 160 Gly Arg Glu Arg Leu Ala Ser ThrAsn Asp Lys Gly Ser Met Ala Met 165 170 175 Glu Ser Ala Lys Glu Thr ArgTyr Cys Ala Val Cys Asn Asp Tyr Ala 180 185 190 Ser Gly Tyr His Tyr GlyVal Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 205 Phe Lys Arg Ser IleGln Gly His Asn Asp Tyr Met Cys Pro Ala Thr 210 215 220 Asn Gln Cys ThrIle Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys 225 230 235 240 Arg LeuArg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg 245 250 255 LysAsp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp 260 265 270Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala 275 280285 Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn 290295 300 Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu305 310 315 320 Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro ThrArg Pro 325 330 335 Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn LeuAla Asp Arg 340 345 350 Glu Leu Val His Met Ile Asn Trp Ala Lys Arg ValPro Gly Phe Val 355 360 365 Asp Leu Thr Leu His Asp Gln Val His Leu LeuGlu Cys Ala Trp Leu 370 375 380 Glu Ile Leu Met Ile Asp Leu Val Trp ArgSer Met Glu His Pro Gly 385 390 395 400 Lys Leu Leu Phe Ala Pro Asn LeuLeu Leu Asp Arg Asn Gln Gly Lys 405 410 415 Cys Val Glu Gly Met Val GluIle Phe Asp Met Leu Leu Ala Thr Ser 420 425 430 Ser Arg Phe Arg Met MetAsn Leu Gln Gly Glu Glu Phe Val Cys Leu 435 440 445 Lys Ser Ile Ile LeuLeu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 450 455 460 Thr Leu Lys SerLeu Glu Glu Lys Asp His Ile His Arg Val Leu Asp 465 470 475 480 Lys IleThr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr 485 490 495 LeuGln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser 500 505 510His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met 515 520525 Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu 530535 540 Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val545 550 555 560 Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser ThrSer Ser 565 570 575 His Pro Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala GluGly Phe Pro 580 585 590 Ala Thr Val 595 11 35 DNA Artificial SequenceDescription of Artificial SequenceDesigned oligonucleotide primer forPCR 11 cctgcgggga cacggtctgc accctgcccg cggcc 35 12 35 DNA ArtificialSequence Description of Artificial SequenceDesigned oligonucleotideprimer for PCR 12 cagggagctc tcagactgtg gcagggaaac cctct 35 13 49 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide for mutagenesis 13 atgatcaaac gctctaagag gaacagcctggccttgtccc tgacggccg 49 14 49 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide for mutagenesis 14ggacaaggcc aggctgttcc tcttagagcg tttgatcatg agcgggctt 49 15 49 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide for mutagenesis 15 aagaacagcc tggccttgtt cctgacggccgaccagatgg tcagtgcct 49 16 49 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide for mutagenesis 16catctggtcg gccgtcagga acaaggccag gctgttcttc ttagagcgt 49 17 44 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide for mutagenesis 17 gctagagatc ctgatgattg atctcgtctggcgctccatg gagc 44 18 44 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide for mutagenesis 18gctccatgga gcgccagacg agatcaatca tcaggatctc tagc 44 19 49 DNA ArtificialSequence Description of Artificial SequenceDesigned oligonucleotide formutagenesis 19 tggtctcgtc tggcgctccg tggagcaccc agggaagcta ctgtttgct 4920 49 DNA Artificial Sequence Description of Artificial SequenceDesignedoligonucleotide for mutagenesis 20 agcaaacagt agcttccctg ggtgctccacggagcgccag acgagacca 49 21 49 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide for mutagenesis 21ctcttggaca ggaaccaggt aaaatgtgta gagggcatgg tggagatct 49 22 49 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide for mutagenesis 22 catgccctct acacatttta cctggttcctgtccaagagc aagttagga 49 23 49 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide for mutagenesis 23ccacctgatg gccaaggcag tcctgaccct gcagcagcag cagcagcac 49 24 49 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide for mutagenesis 24 ggtgctgctg ctgcagggtc aggactgccttggccatcag gtggatcaa 49 25 44 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide for mutagenesis 25atctgtacag catgaagtgc gagaacgtgg tgcccctcta tgac 44 26 44 DNA ArtificialSequence Description of Artificial SequenceDesigned oligonucleotide formutagenesis 26 gtcatagagg ggcaccacgt tctcgcactt catgctgtac agat 44 27 45DNA Artificial Sequence Description of Artificial SequenceDesignedoligonucleotide for mutagenesis 27 gcgggctcta cttcatcgca tcccttgcaaaagtattaca tcacg 45 28 45 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide for mutagenesis 28cgtgatgtaa tacttttgca agggatgcga tgaagtagag cccgc 45 29 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 29 tggagacatg agagctgcca 20 30 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 30 acctttggcc aagcccgctc 20 31 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 31 ccgctcatga tcaaacgctc 20 32 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 32 agggcagggg tgaagtgggg 20 33 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 33 ccaacctttg gccaagcccg 20 34 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 34 tactcggaat agagtatggg 20 35 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 35 ggctcagcat ccaacaaggc 20 36 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 36 tgaccatctg gtcggccgtc 20 37 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 37 aagggtctgg taggatcata 20 38 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 38 catctggtcg gccgtcaggg 20 39 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 39 agagctgcca acctttggcc 20 40 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 40 aagcccgctc atgatcaaac 20 41 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 41 gctctaagaa gaacagcctg 20 42 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 42 agacatgaga gctgccaacc 20 43 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 43 ggccaagccc gctcatgatc 20 44 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 44 agcttcactg aagggtctgg 20 45 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 45 taggatcata ctcggaatag 20 46 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 46 agtatggggg gctcagcatc 20 47 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 47 caacaaggca ctgaccatct 20 48 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 48 aagggtctgg taggatcata 20 49 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 49 gagctggttc acatgatcaa 20 50 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 50 tgggcgaaga gggtgccagg 20 51 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 51 ttgtggattt gaccctccat 20 52 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 52 atcaggtcca ccttctagaa 20 53 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 53 gtgcctggct agagatcctg 20 54 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 54 tgggtgctcc atggagcgcc 20 55 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 55 ttaggagcaa acagtagctt 20 56 19 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 56 tggttcctgt ccaagagca 19 57 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 57 tccaccatgc cctctacaca 20 58 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 58 agccagcagc atgtcgaaga 20 59 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 59 ggctttggtg atttgaccct 20 60 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 60 ccatgatcag gtccaccttc 20 61 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 61 tagaatgtgc ctggctagag 20 62 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 62 gctggttcac atgatcaact 20 63 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 63 gccaggcttt gtggatttga 20 64 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 64 atgatgtagc cagcaggtcg 20 65 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 65 gccctctaca cattttccct 20 66 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 66 aagagcaagt taggagcaaa 20 67 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 67 ggatcaaagt gtctgtgatc 20 68 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 68 tctccaccat gccctctaca 20 69 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 69 ggtctcgtct ggcgctccat 20 70 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 70 ggagcaccca gtgaagctac 20 71 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 71 tgtttgctcc taacttggac 20 72 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 72 tcctgatgat tggtctcgtc 20 73 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 73 cacccagtga agctactgtt 20 74 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 74 ttcatcatgc ggaaccgaga 20 75 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 75 gatgtagcca gcagcatgtc 20 76 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 76 aagatctcca ccatgcccct 20 77 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 77 ctccaccatg cccctctaca 20 78 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 78 atgtcgaaga tctccaccat 20 79 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 79 agagaaggac catatccacc 20 80 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 80 gagtcctgga caagatcaca 20 81 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 81 gacactttga tccacctgat 20 82 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 82 ccctgaagtc tctggaagag 20 83 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 83 acaagatcac agacactttg 20 84 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 84 tgtgcctgat gtgggagagg 20 85 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 85 atgaggagga gctgggccag 20 86 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 86 ccgctggtgc tgctgctgca 20 87 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 87 gctgggccag ccgctggtgc 20 88 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 88 cctttgttac tcatgtgcct 20 89 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 89 aggcacatga gtaacaaagg 20 90 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 90 catggagcat ctgtacagca 20 91 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 91 ttgtggattt gaccctccat 20 92 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 92 ccaccgagtc ctggacaaga 20 93 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 93 ggccaaggca ggcctgaccc 20 94 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 94 gtaggcggtg ggcgtccagc 20 95 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 95 atctccagca gcaggtcata 20 96 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 96 cagcaggtca tagaggggca 20 97 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 97 cagtggccaa gtggctttgg 20 98 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 98 ccacggctag tgggcgcatg 20 99 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 99 aagtgcaaga acgtggtgcc 20 100 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 100 tctatgacct gctgctggag 20 101 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 101 tgctggacgc ccaccgccta 20 102 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 102 atgcgcccac tagccgtgga 20 103 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 103 gcatccgtgg aggagacgga 20 104 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 104 tcccccgtga tgtaatactt 20 105 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 105 tctgcctccc ccgtgatgta 20 106 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 106 aaaccctctg cctcccccgt 20 107 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 107 gtggcaggga aaccctctgc 20 108 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 108 actgtggcag ggaaaccctc 20 109 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 109 atatgtgtcc agccaccaac 20 110 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 110 tatctgaacc gtgtgggagc 20 111 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide probe for Southern hybridization 111 tgatcaaacgctctaagaag 20 112 20 DNA Artificial Sequence Description of ArtificialSequenceDesigned oligonucleotide probe for Southern hybridization 112caaacgctct aagaagaaca 20 113 20 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide probe for Southernhybridization 113 cgctctaaga agaacagcct 20 114 20 DNA ArtificialSequence Description of Artificial SequenceDesigned oligonucleotideprobe for Southern hybridization 114 ctaagaagaa cagcctggcc 20 115 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide probe for Southern hybridization 115 gaagaacagcctggccttgt 20 116 20 DNA Artificial Sequence Description of ArtificialSequenceDesigned oligonucleotide probe for Southern hybridization 116agaacagcct ggccttgtcc 20 117 20 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide probe for Southernhybridization 117 cagcctggcc ttgtccctga 20 118 20 DNA ArtificialSequence Description of Artificial SequenceDesigned oligonucleotideprobe for Southern hybridization 118 ctggccttgt ccctgacggc 20 119 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide probe for Southern hybridization 119 ccttgtccctgacggccgac 20 120 20 DNA Artificial Sequence Description of ArtificialSequenceDesigned oligonucleotide probe for Southern hybridization 120gtccctgacg gccgaccaga 20 121 20 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide probe for Southernhybridization 121 gagatcctga tgattggtct 20 122 20 DNA ArtificialSequence Description of Artificial SequenceDesigned oligonucleotideprobe for Southern hybridization 122 atcctgatga ttggtctcgt 20 123 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide probe for Southern hybridization 123 ctgatgattggtctcgtctg 20 124 20 DNA Artificial Sequence Description of ArtificialSequenceDesigned oligonucleotide probe for Southern hybridization 124atgattggtc tcgtctggcg 20 125 20 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide probe for Southernhybridization 125 attggtctcg tctggcgctc 20 126 20 DNA ArtificialSequence Description of Artificial SequenceDesigned oligonucleotideprobe for Southern hybridization 126 gtctcgtctg gcgctccatg 20 127 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide probe for Southern hybridization 127 acgtctggcgctccatggag 20 128 20 DNA Artificial Sequence Description of ArtificialSequenceDesigned oligonucleotide probe for Southern hybridization 128ggcgctccat ggaggcaccc 20 129 21 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide probe for Southernhybridization 129 gctccatgga ggcacccagg g 21 130 20 DNA ArtificialSequence Description of Artificial SequenceDesigned oligonucleotideprobe for Southern hybridization 130 catggaggca cccagggaag 20 131 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide probe for Southern hybridization 131 tcttggacaggaaccaggga 20 132 20 DNA Artificial Sequence Description of ArtificialSequenceDesigned oligonucleotide probe for Southern hybridization 132ggacaggaac cagggaaaat 20 133 20 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide probe for Southernhybridization 133 aggaaccagg gaaaatgtgt 20 134 20 DNA ArtificialSequence Description of Artificial SequenceDesigned oligonucleotideprobe for Southern hybridization 134 accagggaaa atgtgtagag 20 135 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide probe for Southern hybridization 135 gggaaaatgtgtagagggca 20 136 20 DNA Artificial Sequence Description of ArtificialSequenceDesigned oligonucleotide probe for Southern hybridization 136acctgatggc caaggcaggc 20 137 20 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide probe for Southernhybridization 137 gatggccaag gcaggcctga 20 138 20 DNA ArtificialSequence Description of Artificial SequenceDesigned oligonucleotideprobe for Southern hybridization 138 gccaaggcag gcctgaccct 20 139 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide probe for Southern hybridization 139 aggcaggcctgaccctgcag 20 140 20 DNA Artificial Sequence Description of ArtificialSequenceDesigned oligonucleotide probe for Southern hybridization 140aggcctgacc ctgcagcagc 20 141 20 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide probe for Southernhybridization 141 ctgtacagca tgaagtgcaa 20 142 20 DNA ArtificialSequence Description of Artificial SequenceDesigned oligonucleotideprobe for Southern hybridization 142 acagcatgaa gtgcaagaac 20 143 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide probe for Southern hybridization 143 atgaagtgcaagaacgtggt 20 144 20 DNA Artificial Sequence Description of ArtificialSequenceDesigned oligonucleotide probe for Southern hybridization 144agtgcaagaa cgtggtgccc 20 145 20 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide probe for Southernhybridization 145 caagaacgtg gtgcccctct 20 146 20 DNA ArtificialSequence Description of Artificial SequenceDesigned oligonucleotideprobe for Southern hybridization 146 gctctacttc atcgcattcc 20 147 20 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide probe for Southern hybridization 147 ctacttcatcgcattccttg 20 148 20 DNA Artificial Sequence Description of ArtificialSequenceDesigned oligonucleotide probe for Southern hybridization 148cttcatcgca ttccttgcaa 20 149 20 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide probe for Southernhybridization 149 catcgcattc cttgcaaaag 20 150 20 DNA ArtificialSequence Description of Artificial SequenceDesigned oligonucleotideprobe for Southern hybridization 150 cgcattcctt gcaaaagtat 20 151 40 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for PCR 151 cccagccacc atgaccatga ccctccacaccaaagcatct 40 152 21 DNA Artificial Sequence Description of ArtificialSequenceDesigned oligonucleotide primer for mutagenesis 152 caggctgttcctcttagagc g 21 153 31 DNA Artificial Sequence Description of ArtificialSequenceDesigned oligonucleotide primer for mutagenesis 153 tggtcggccgtcaggaacaa ggccaggctg t 31 154 21 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide primer for mutagenesis 154gggtgctcca cggagcgcca g 21 155 31 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide primer for mutagenesis 155ccctctacac attttacctg gttcctgtcc a 31 156 31 DNA Artificial SequenceDescription of Artificial SequenceDesigned oligonucleotide primer formutagenesis 156 tgctgcaggg tcaggactgc cttggccatc a 31 157 23 DNAArtificial Sequence Description of Artificial SequenceDesignedoligonucleotide primer for mutagenesis 157 caaagcctgg ctccctcttc gcc 23158 24 DNA Artificial Sequence Description of ArtificialSequenceDesigned oligonucleotide primer for PCR 158 ggaatgatgaaaggtgggat acga 24 159 25 DNA Artificial Sequence Description ofArtificial SequenceDesigned oligonucleotide primer for PCR 159aatttatgct acaacaaggc aaggc 25 160 24 DNA Artificial SequenceDescription of Artificial Sequence Designed oligonucleotide primer forPCR 160 ggagtggcac cttccagggt caag 24 161 33 DNA Artificial SequenceDescription of Artificial Sequence Designed oligonucleotide forsynthesis 161 tcgacaaagt caggtcacag tgacctgatc aag 33 162 52 DNAArtificial Sequence Description of Artificial Sequence Designedoligonucleotide for synthesis 162 gatctcgact ataaagaggg caggctgtcctctaagcgtc accacgactt ca 52 163 52 DNA Artificial Sequence Descriptionof Artificial Sequence Designed oligonucleotide for synthesis 163agcttgaagt cgtggtgacg cttagaggac agcctgccct ctttatagtc ga 52 164 26 DNAArtificial Sequence Description of Artificial Sequence Designedoligonucleotide primer for PCR 164 gtggagacat gagagctgcc aacctt 26 16540 DNA Artificial Sequence Description of Artificial Sequence Designedoligonucleotide primer for PCR 165 gaccatctgg tcggccgtca gggacaaggccaggctaggc 40 166 40 DNA Artificial Sequence Description of ArtificialSequence Designed oligonucleotide primer for PCR 166 gctcatgatcaaacgctcta agaagaacag cctgcctggg 40 167 24 DNA Artificial SequenceDescription of Artificial Sequence Designed oligonucleotide primer forPCR 167 aatagagtat ggggggctca gcat 24 168 25 DNA Artificial SequenceDescription of Artificial Sequence Designed oligonucleotide primer forPCR 168 ggtccacctt ctagaatgtg cctgg 25 169 39 DNA Artificial SequenceDescription of Artificial Sequence Designed oligonucleotide primer forPCR 169 gcaagttagg agcaaacagt agcttccctg ggtggtgca 39 170 25 DNAArtificial Sequence Description of Artificial Sequence Designedoligonucleotide primer for PCR 170 ccatggagca gggagtgaag ctact 25 171 40DNA Artificial Sequence Description of Artificial Sequence Designedoligonucleotide primer for PCR 171 cagcatgtcg aagatctcca ccatgccctctacacatggt 40 172 24 DNA Artificial Sequence Description of ArtificialSequence Designed oligonucleotide primer for PCR 172 gagtcctggacaagatcaca gaca 24 173 24 DNA Artificial Sequence Description ofArtificial Sequence Designed oligonucleotide primer for PCR 173tgctgtacag atgctccatg cctt 24 174 24 DNA Artificial Sequence Descriptionof Artificial Sequence Designed oligonucleotide primer for PCR 174ctctcccaca tcaggcacat gagt 24 175 40 DNA Artificial Sequence Descriptionof Artificial Sequence Designed oligonucleotide primer for PCR 175agcatctcca gcagcaggtc atagaggggc accacgagct 40 176 35 DNA ArtificialSequence Description of Artificial Sequence Designed oligonucleotideprimer for PCR 176 gaggcggggt aagggaagta ggtggaagat tcagc 35 177 35 DNAArtificial Sequence Description of Artificial Sequence Designedoligonucleotide primer for PCR 177 gggtggggaa atagggtttc caatgcttcactggg 35 178 40 DNA Artificial Sequence Description of ArtificialSequence Designed oligonucleotide primer for PCR 178 cccagccaccatggaagtgc agttagggct gggaagggtc 40 179 35 DNA Artificial SequenceDescription of Artificial Sequence Designed oligonucleotide primer forPCR 179 gggtggggaa atagggtttc caatgcttca ctggg 35 180 30 DNA ArtificialSequence Description of Artificial Sequence Designed oligonucleotideprimer for PCR 180 gcgttcacaa gctaagttgt ttatctcggc 30 181 30 DNAArtificial Sequence Description of Artificial Sequence Designedoligonucleotide primer for PCR 181 taaatttcac catctactct cccatcactg 30182 38 DNA Artificial Sequence Description of Artificial SequenceDesigned oligonucleotide primer for PCR 182 ccaccatgga ctccaaagaatcattaactc ctggtaga 38 183 35 DNA Artificial Sequence Description ofArtificial Sequence Designed oligonucleotide primer for PCR 183gcagtcactt ttgatgaaac agaagttttt tgata 35 184 29 DNA Artificial SequenceDescription of Artificial Sequence Designed oligonucleotide primer forPCR 184 ccgacccagg aggtggagat ccctccggt 29 185 24 DNA ArtificialSequence Description of Artificial Sequence Designed oligonucleotideprimer for PCR 185 ccacaaaatt taattcttta aaag 24 186 35 DNA ArtificialSequence Description of Artificial Sequence Designed oligonucleotideprimer for PCR 186 ccaccatgac tgagctgaag gcaaagggtc cccgg 35 187 35 DNAArtificial Sequence Description of Artificial Sequence Designedoligonucleotide primer for PCR 187 cattcacttt ttatgaaaga gaaggggtttcacca 35 188 30 DNA Artificial Sequence Description of ArtificialSequence Designed oligonucleotide primer for PCR 188 gcactcgctggcctggatgt ggttggattt 30 189 30 DNA Artificial Sequence Description ofArtificial Sequence Designed oligonucleotide primer for PCR 189ttcagactgc tctggtctcg ccaaatccac 30 190 34 DNA Artificial SequenceDescription of Artificial Sequence Designed oligonucleotide primer forPCR 190 ccaccatgga gaccaaaggc taccacagtc tccc 34 191 34 DNA ArtificialSequence Description of Artificial Sequence Designed oligonucleotideprimer for PCR 191 cagtcacttc cggtggaagt agagcggctt ggcg 34 192 35 DNAArtificial Sequence Description of Artificial Sequence Designedoligonucleotide primer for PCR 192 ttgagttact gagtccgatg aatgtgcttgctctg 35 193 35 DNA Artificial Sequence Description of ArtificialSequence Designed oligonucleotide primer for PCR 193 aaatgagggaccacacagca gaaagatgaa gccca 35 194 55 DNA Artificial SequenceDescription of Artificial Sequence Designed oligonucleotide primer forPCR 194 gccgcggccg cccagccacc atggatataa aaaactcacc atctagcctt aattc 55195 41 DNA Artificial Sequence Description of Artificial SequenceDesigned oligonucleotide primer for PCR 195 gggtctagaa atgagggaccacacagcaga aagatgaagc c 41 196 37 DNA Artificial Sequence Description ofArtificial Sequence Designed oligonucleotide primer for PCR 196tggaattgaa gtgaatggaa cagaagccaa gcaaggt 37 197 35 DNA ArtificialSequence Description of Artificial Sequence Designed oligonucleotideprimer for PCR 197 tggccgcctg aggctttaga cttcctgatc ctcaa 35 198 40 DNAArtificial Sequence Description of Artificial Sequence Designedoligonucleotide primer for PCR 198 cccagccacc atggaacaga agccaagcaaggtggagtgt 40 199 35 DNA Artificial Sequence Description of ArtificialSequence Designed oligonucleotide primer for PCR 199 tggccgcctgaggctttaga cttcctgatc ctcaa 35 200 35 DNA Artificial SequenceDescription of Artificial Sequence Designed oligonucleotide primer forPCR 200 ttactaacct ataaccccca acagtatgac agaaa 35 201 35 DNA ArtificialSequence Description of Artificial Sequence Designed oligonucleotideprimer for PCR 201 cagtctaatc ctcgaacact tccaggaaca aaggg 35 202 40 DNAArtificial Sequence Description of Artificial Sequence Designedoligonucleotide primer for PCR 202 cccagccacc atgacagaaa atggccttacagcttgggac 40 203 35 DNA Artificial Sequence Description of ArtificialSequence Designed oligonucleotide primer for PCR 203 cagtctaatcctcgaacact tccaggaaca aaggg 35 204 20 DNA Artificial SequenceDescription of Artificial Sequence Designed oligonucleotide forsynthesis 204 tcaggtcatt ccaggtcatg 20 205 30 DNA Artificial SequenceDescription of Artificial Sequence Designed oligonucleotide primer forPCR 205 agaagccttt gggtctgaag tgtctgtgag 30 206 30 DNA ArtificialSequence Description of Artificial Sequence Designed oligonucleotideprimer for PCR 206 atggctgagg tctcaaggga ccggggaaaa 30 207 33 DNAArtificial Sequence Description of Artificial Sequence Designedoligonucleotide primer for PCR 207 ccaccatgga ggcaatggcg gccagcactt ccc33 208 32 DNA Artificial Sequence Description of Artificial SequenceDesigned oligonucleotide primer for PCR 208 tagtcaggag atctcattgccaaacacttc ga 32 209 19 DNA Artificial Sequence Description ofArtificial Sequence Designed oligonucleotide for synthesis 209tcaggtcaca gaggtcatg 19 210 30 DNA Artificial Sequence Description ofArtificial Sequence Designed oligonucleotide primer for PCR 210ggattgatct tttgctagat agagacaaaa 30 211 33 DNA Artificial SequenceDescription of Artificial Sequence Designed oligonucleotide primer forPCR 211 ctagtacaag tccttgtaga tctcctgcag gag 33 212 33 DNA ArtificialSequence Description of Artificial Sequence Designed oligonucleotideprimer for PCR 212 ccaccatggg tgaaactctg ggagattctc cta 33 213 17 DNAArtificial Sequence Description of Artificial Sequence Designedoligonucleotide for synthesis 213 tcaggtcaca ggtcatg 17

1. An artificial cell comprising: (i) a chromosome which comprises areporter gene, wherein the reporter gene comprises an ERE, a TATAsequence and a reporter sequence naturally foreign to the ERE; and (ii)one or more factors selected from the following (ii-a) and (ii-b):(ii-a) a mutant ERα which has an activity for transactivation of thereporter gene, wherein in the presence of a partial anti-estrogen and E2the activity is higher than that of a normal ERα in the presence of thepartial anti-estrogen and E2 or in the presence of a partialanti-estrogen the activity is higher than that of a normal ERα in thepresence of the partial anti-estrogen; and (ii-b) a gene which encodesthe mutant ERα.
 2. The artificial cell according to claim 1, wherein thegene of (ii-b) is comprised by the chromosome of (i).
 3. The artificialcell according to claim 1, wherein the gene of (ii-b) is comprised by avector.
 4. The artificial cell according to claim 1, wherein the partialanti-estrogen is tamoxifen, raloxifene or 4-hydroxytamoxifen.
 5. Theartificial cell according to claim 1, wherein the activity is also anactivity for transactivation of the reporter gene which is inhibited inthe presence of a pure anti-estrogen.
 6. The artificial cell accordingto claim 1, wherein the mutant ERα has one or more substituted aminoacids which confer the activity.
 7. The artificial cell according toclaim 1, wherein the mutant ERα has one or more substituted amino acidswhich confer the activity at one or more relative positions from 303 to578, wherein the relative positions are based on a homology alignment toan amino acid sequence shown in SEQ ID:
 1. 8. The artificial cellaccording to claim 1, wherein the mutant ERα has one or more substitutedamino acids which confers the activity at one or more relative positionsselected from 303, 309, 390, 396, 415, 494, 531 and 578, wherein therelative positions are based on a homology alignment to an amino acidsequence shown in SEQ ID:
 1. 9. The artificial cell according to claim1, wherein the normal ERα is an ERα having an amino acid sequence shownin SEQ ID:
 1. 10. An artificial cell comprising: a chromosome whichcomprises a reporter gene, wherein the reporter gene comprises an ERE, aTATA sequence and a reporter sequence naturally foreign to the ERE; anda mutant ERα which activates transcription of a gene downstream from anERE while exposed to an anti-estrogen which is not antagonistic to anAF1 region of a normal ERα and is antagonistic to an AF2 region of anormal ERα.
 11. The artificial cell according to claim 10, wherein themutant ERα is one which also activates transcription of the genedownstream from an ERE while bound to E2, wherein the activation is notinhibited by the anti-estrogen which is not antagonistic to an AF1region of a normal ERα and is antagonistic to an AF2 region of a normalERα.
 12. An isolated mutant ERα having: an activity for transactivationof a reporter gene, the reporter gene comprising an ERE, a TATA sequenceand a reporter sequence naturally foreign to the ERE, wherein in thepresence of a partial anti-estrogen and E2 the activity is higher thanthat of a normal ERα in the presence of the partial anti-estrogen andE2, or in the presence of a partial anti-estrogen the activity is higherthan that of a normal ERα in the presence of the partial anti-estrogen;and an amino acid sequence of an ERα comprising one or more substitutedamino acids at one or more relative positions selected from 303, 309,390, 396, 494 and 578, or two or more substituted amino acids at two ormore relative positions selected from 303, 309, 390, 396, 415, 494, 531and 578, wherein the relative positions are based on a homologyalignment to an amino acid sequence shown in SEQ ID:
 1. 13. The isolatedmutant ERα according to claim 12, wherein the substituted amino acid isan arginine at relative position 303, a phenylalanine at relativeposition 309, a asparaginic acid at relative position 390, a valine atrelative position 396, a valine at relative position 494 or a proline ata relative position 578, wherein the relative positions are based on ahomology alignment to an amino acid sequence shown in SEQ ID:
 1. 14. Theisolated mutant ERα according to claim 12, which is derived from anormal ERα comprising a lysine at relative position 303, a serine atrelative position 309, a glycine at relative position 390, a methionineat relative position 396, a glycine at relative position 494 and a seineat relative position 578, wherein the relative positions are based on ahomology alignment to an amino acid sequence shown in SEQ ID:
 1. 15. Anisolated mutant ERα having an amino acid sequence shown in SEQ ID: 2.16. An isolated mutant ERα having an amino acid sequence shown in SEQID:
 3. 17. An isolated mutant ERα having an amino acid sequence shown inSEQ ID:
 4. 18. An isolated mutant ERα having an amino acid sequenceshown in SEQ ID:
 5. 19. An isolated mutant ERα having an amino acidsequence shown in SEQ ID:
 7. 20. An isolated mutant ERα having an aminoacid sequence shown in SEQ ID:
 9. 21. An isolated mutant ERα having anamino acid sequence shown in SEQ ID:
 10. 22. An isolated polynucleotideencoding the mutant ERα of claim
 12. 23. An isolated polynucleotideencoding the mutant ERα of claim
 15. 24. An isolated polynucleotideencoding the mutant ERα of claim
 16. 25. An isolated polynucleotideencoding the mutant ERα of claim
 17. 26. An isolated polynucleotideencoding the mutant ERα of claim
 18. 27. An isolated polynucleotideencoding the mutant ERα of claim
 19. 28. An isolated polynucleotideencoding the mutant ERα of claim
 20. 29. An isolated polynucleotideencoding the mutant ERα of claim
 21. 30. A vector comprising thepolynucleotide of claim
 22. 31. A virus comprising the vector of claim30.
 32. A method for quantitatively analyzing an activity fortransactivation of a reporter gene by a test ERα, the method comprising:exposing an artificial cell with a ligand, the artificial cellcomprising the test ERα and a chromosome which comprises the reportergene wherein the reporter gene comprises an ERE, a TATA sequence and areporter sequence naturally foreign to the ERE; and measuring atransactivation amount of the reporter gene by the test ERα.
 33. Themethod according to claim 32, wherein the ligand is a partialanti-estrogen.
 34. The method according to claim 32, wherein the ligandis tamoxifen, raloxifene or 4-hydroxy-tamoxifen.
 35. The methodaccording to claim 32, wherein the ligand is an anti-estrogen which isnot antagonistic to an AF1 region and is antagonistic to an AF2 region.36. A method for screening a test ligand dependent transcriptionalfactor, the method comprising: exposing an artificial cell with aligand, the artificial cell comprising a test ligand dependenttranscriptional factor and a chromosome which comprises the reportergene, wherein the reporter gene comprises a receptor responsive sequencecognate with the test ligand dependent transcriptional factor, a TATAsequence and a reporter sequence naturally foreign to the receptorresponsive sequence; measuring a transactivation amount of the reportergene by the test ligand dependent transcriptional factor; comparing thetransactivation amount of the reporter gene by the test ligand dependenttranscriptional factor to a transactivation amount of the reporter geneby a standard; and selecting the test ligand dependent transcriptionalfactor wherein the transactivation amount of the reporter gene by thetest transactivational amount is different than the transactivationamount of the reporter gene by the standard.
 37. The method according toclaim 36, wherein the test ligand dependent transcriptional factor is amutant ligand dependent transcriptional factor and the standard is anormal test ligand dependent transcriptional factor.
 38. The methodaccording to claim 36, wherein the test ligand dependent transcriptionalfactor is a test ERα, a test ERβ, a test AR, a test GR, a test PR, atest MR, a test receptor naturally having a lipophilic vitamin as aligand, a test PPAR or a test TR.
 39. A method for screening a test ERα,the method comprising: exposing an artificial cell with a ligand, theartificial cell comprising the test ERα and a chromosome which comprisesthe reporter gene, wherein the reporter gene comprises an ERE, a TATAsequence and a repolter sequence naturally foreign the ERE; measuring atransactivation amount of the reporter gene by the test ERα; comparingthe transactivation amount of the reporter gene by the test ERα to atransactivational amount of the reporter gene by a standard; andselecting the test ERα wherein the transactivation amount of thereporter gene by the test ERα is different than the transactivationamount of the reporter gene by the standard.
 40. The method according toclaim 39, wherein the standard is a normal ERα, a normal ERα having anamino acid sequence shown in SEQ ID: 1 or an ERα which phenotype isknown.
 41. The method according to claim 39, wherein the ligand is apartial anti-estrogen.
 42. The method according to claim 39, wherein theligand is tamoxifen, raloxifene or 4-hydroxy-tamoxifen.
 43. A method forevaluating an activity for transactivation of a reporter gene by a testERα, the method comprising: exposing an artificial cell with a ligand,the artificial cell comprising the test ERα and a chromosome whichcomprises the reporter gene, wherein the reporter gene comprises an ERE,a TATA sequence and a reporter sequence naturally foreign the ERE;measuring a transactivation amount of the reporter gene by the test ERα;and comparing the transactivation amount of the reporter gene by thetest ERα to a transactivational amount of the reporter gene by astandard.
 44. The method according to claim 43, wherein the standard isa normal ERα, a normal ERα having an amino acid sequence shown in SEQID: 1 or an ERα which phenotype is known.
 45. The method according toclaim 43, wherein the ligand is a partial anti-estrogen.
 46. The methodaccording to claim 43, wherein the ligand is tamoxifen, raloxifene4-hydroxy-tamoxifen.
 47. A method for screening a compound useful fortreating a disorder of a mutant ERα, the method comprising: exposing theartificial cell of claim 1 with a test compound; measuring atransactivation amount of the reporter gene of the artificial cell. 48.A pharmaceutical agent useful for treating a disorder of a mutant ERα,the agent comprising as an active ingredient a compound screened by themethod of claim
 46. 49. Use of the mutant ERα of claim 12 for a receptorbinding assay.
 50. A method for diagnosing a genotype of apolynucleotide encoding a test ERα, the method comprising: searching ina nucleotide sequence of the polynucleotide encoding the test ERα forone or more valiant codons which encode one or more substituted aminoacids in the test ERα wherein the one or more substituted amino acidsconfer an activity for transactivation of a reporter gene, in which inthe presence of a partial anti-estrogen and E2 the activity is higherthan that of a normal ERα in the presence of the partial anti-estrogenand E2 or which in the presence of the partial anti-estrogen theactivity is higher than that of a normal ERα in the presence of thepartial anti-estrogen, and in which a chromosome comprises the reportergene, the reporter gene comprising an ERE, a TATA sequence and areporter sequence naturally foreign the ERE; and determining themutation in the nucleotide sequence of the one or more variant codons bycomparing the nucleotide sequence of the one or more variant codons to anucleotide sequence of one or more corresponding codons in a nucleotidesequence encoding a standard.
 51. The method according to claim 50,wherein the nucleotide sequence of the polynucleotide encoding the testERα is searched at one or more variant codons which encode an amino acidat one or more relative positions selected from 303, 309, 390, 396, 494and 578, or an amino acid at two or more relative positions selectedfrom 303, 309, 390, 396, 415, 494, 531 and 578, wherein the relativepositions are based on a homology alignment to an amino acid sequenceshown in SEQ ID:
 1. 52. The method according to claim 50, wherein thestandard is a normal ERα, a normal ERα having an amino acid sequenceshown in SEQ ID: 1 or an ERα which phenotype is known.
 53. A method fordiagnosing a phenotype of a test ERα, the method comprising: searchingin an amino acid sequence of the test ERα for one or more substitutedamino acids in the test ERα, wherein the one or more substituted aminoacids confer an activity for transactivation of a reporter gene, inwhich in the presence of a partial anti-estrogen and E2 the activity ishigher than that of a normal ERα in the presence of the partialanti-estrogen and E2 or which in the presence of a partial anti-estrogenthe activity is higher than that of a normal ERα in the presence of thepartial anti-estrogen, and in which a chromosome comprises the reportergene, the reporter gene comprising an ERE, a TATA sequence and areporter sequence naturally foreign to the ERE; and determining themutation in the amino acid sequence of the test ERα by comparing theamino acid sequence of the test ERα to an amino acid sequence of astandard.
 54. The method according to claim 53, wherein the an aminoacid sequence of the test ERα is searched at one or more relativepositions selected from 303, 309, 390, 396, 494 and 578, or at two ormore relative positions selected from 303, 309, 390, 396, 415, 494, 531and 578, wherein the relative positions are based on a homologyalignment to an amino acid sequence shown in SEQ ID:
 1. 57. The methodaccording to claim 53, wherein the standard is a normal ERα, a normalERα having an amino acid sequence shown in SEQ ID: 1 or an ERα whichphenotype is known.